Documentos de Académico
Documentos de Profesional
Documentos de Cultura
TESIS DOCTORAL
Granada, 2012
Editor: Editorial de la Universidad de Granada
Autor: Beatriz Estrada Velasco
D.L.: GR 1058-2013
ISBN: 978-84-9028-489-6
Characterization of the adaptation of mycorrhizal
fungi to environmnets affected by drought and salinity
and their implications in the improvement of plant
tolerance to such environments
DOCTORAL THESIS
Granada, 2012
UNIVERSIDAD DE GRANADA
FACULTAD DE CIENCIAS
Departamento de Fisiología Vegetal
Resumen
Abstract 48
Introduction 48
Material and Methods 50
Results 52
Discussion 56
Conclusions 59
References 60
CAPÍTULO 2: Diversispora clara (Glomeromycetes) – una nueva especie de
dunas salinas del Parque Natural Cabo de Gata (España) 69
Resumen
Abstract 72
Introduction 72
Material and Methods 72
Results 74
Discussion 76
References 79
Resumen
Abstract 88
Introduction 89
Material and Methods 91
Results 97
Discussion 105
References 110
Resumen
CHAPTER 4: Arbuscular mycorrhizal fungi native from a Mediterranean
saline area enhance maize plants tolerance to salinity through improved ion
homeostasis 123
Abstract 124
Introduction 124
Material and Methods 127
Results 131
Discussion 139
References 143
Resumen
Abstract 154
Introduction 154
Material and Methods 156
Results 161
Discussion 167
References 170
Resumen
CHAPTER 6 Importance of native arbuscular mycorrhizal inoculation in
the halophyte Asteriscus maritimus for successful establishment and growth
under saline conditions 181
Abstract 182
Introduction 182
Material and Methods 184
Results 190
Discussion 198
References 202
CONCLUSIONES 211
CONCLUSIONS 213
NATURALEZA, INTERÉS Y OBJETIVOS
INTEREST OF THE STUDY AND AIMS
Naturalza, interés y objetivos
Una de las estrategias que han desarrollado las plantas para tolerar el estrés salino
se basa en su asociación con microorganismos rizosféricos (hongos y bacterias). En
particular, un componente clave de la microbiota del suelo, son los hongos formadores
de micorrizas arbusculares (MA), que se postulan como uno de los factores más
influyentes en el mantenimiento de la variabilidad, estabilidad, diversidad y
3
Naturaleza, interés y objetivos
4
Naturalza, interés y objetivos
3. Determinar los mecanismos fisiológicos por lo que los hongos MA nativos del
Parque incrementan la tolerancia al estrés salino de plantas glicófitas de interés
agronómico y halófitas de interés en revegetación.
5
Interest of the study and aims
The stress is mainly due to climatic conditions (seasonality with hot and dry
season together) that produces highly unpredictable rainfall patterns that can lead to a
hydric limitation, soils poor in nutrients (nitrogen and phosphorus) and soils with high
salinity. The salinity problem is accentuated by the limited annual rainfall which is
insufficient to transport the soluble salts out of the rhizosphere of the plants. Moreover,
the characteristics of these regions: high levels of evaporation and solar radiation,
contribute to further increase the salinity of soils.
One strategy that plants have developed to tolerate salt stress is based on its
association with rhizospheric microorganisms (fungi and bacteria). In particular, a key
component of soil microbiota, the arbuscular mycorrhizal fungi (AMF), are postulated
as one of the most influential factors in the maintenance of variability, stability,
diversity and productivity of the vegetation cover. The AM roots explore greater
volumes of soil at greater depths and distances to supply water and nutrients to the host
plant than roots of non-mycorrhizal plants. In fact, Mediterranean ecosystems together
with their particular plant species, offer a model of interest to conduct research projects
to study the impact of mycorrhizal associations in the maintenance and development of
these ecosystems.
7
Interest of the study and aims
For all the above reasons, the study of the AMF of arid and semi-arid salinized
ecosystems is critical because they are important potential sources of AMF inocula
adapted to these conditions. The AMF isolated in Mediterranean ecosystems affected by
salinity might be used as inoculum to achieve plant establishment under natural
conditions of osmotic stress, being especially useful in environmental restoration
practices of degraded ecosystems or under desertification process. The present Doctoral
Thesis was outlined in the context of the conceptual topics above mentioned and it is
part of a research project conducted within the Excellence Project Program in I+D+i of
PIDI (Junta de Andalucía). Although the strategy of I+D+i proposed can be extrapolated
to any ecosystem affected by drought/salinity, the proposal focuses on a defined
scientifically representative sampling area and socioculturally emblematic part of
Andalucía: the Natural Park Cabo de Gata, Almería, with characteristic long sandy
ribbons, salt marshes, dunes and aridity, together with vegetation adapted to the territory
and AMF. Therefore, the aim of the thesis was To investigate the natural diversity of
AMF in the ecosystem, and the physiological and molecular mechanisms that govern
the processes of adaptation of these fungi to salinity and repercussion on plant
tolerance (survival, development and productivity) under salinity conditions.
8
Interest of the study and aims
To achieve this main objective, the following specific objectives were proposed:
1. To analyze the diversity of AMF present in the rhizosphere of the plants in the
dunes and salt marshes of the Park and establish a germplasm bank of AMF
adapted to the ecosystem.
Although the proposed objectives are intended to advance in the knowledge of the
ecology and physiological functioning in the relationship fungus-plant of AMF in arid
ecosystems typical of Mediterranean environments, the research also presents a strong
proposal. The latter is determined by the ability to evaluate the effect of conducted
mycorrhization in the development of both plants for restoration of degraded areas and
agricultural improvement in areas affected by salinity.
9
INTRODUCCIÓN
Introducción
INTRODUCCIÓN
13
Introducción
En las áreas mediterráneas, las lluvias escasas e irregulares, con largos, secos y
calurosos veranos, junto con la presión antropogénica y abandono de suelos agrícolas,
son factores determinantes de la degradación del ecosistema (Barea et al., 2006). En
particular en el sureste de España, esta situación de estrés múltiple está llevando a
desertificación de parte del territorio (Francis and Thornes, 1990; Albaladejo et al.,
1996). donde aproximadamente un 18% del territorio presenta graves procesos de
erosión y desertificación, siendo la cifra en Andalucía del 36%, debido principalmente a
las características accidentales del relieve, con fuertes pendientes y desniveles, que
acentúan el efecto de las precipitaciones (Cornejo, 2006).
14
Introducción
Límites
Límites deldel PN
Reserva terrestre
Reserva
Reserva marina
Reserva
Figura 1. Localización del Parque Natural de Cabo de Gata y sus zonas protegidas
Este enclave es uno de los pocos lugares, en toda Europa, protegido por su carácter
semiárido y estepario. Las condiciones climáticas de sequedad de Cabo de Gata son
semejantes a las que existen en extensos territorios de África del Norte o de Oriente
Medio, lo que identifica este lugar como el enclave más árido de Europa Occidental
(Geiger, 1973). A pesar de ello y de su aparente aspecto desértico encierra formas de
vida animal y vegetal muy peculiares, que han logrado adaptarse a extremas
condiciones de aridez, caracterizada por su elevada insolación media (2960 horas), el
índice de precipitaciones mas bajo de la península y la suavidad de su régimen térmico
15
Introducción
16
Introducción
del suelo provocando una pérdida de cubierta vegetal y microbiota del suelo (Requena
et al., 1996). La microbiota del suelo juega un papel fundamental en la regulación de los
ecosistemas terrestres, influyendo en la productividad, diversidad y estructura de las
comunidades vegetales (van der Heijden et al., 2008). Entre los organismos que habitan
en el suelo cabe destacar por su función ecológica los hongos micorrícico arbusculares
(ver punto 3). El deterioro de los sistemas suelo-planta, en cuanto que afecta a las
relaciones planta-microorganismos, desencadena un círculo vicioso de efectos
negativos. Si no hay plantas, se degrada la vida microbiota del suelo y si no hay
propágulos, cualquier proceso natural o inducido de revegetación presenta problemas
para prosperar adecuadamente (Marulanda, 2006). Por lo tanto para evitar la
degradación de este tipo de ecosistemas, que por sus condiciones extremas son más
sensibles, es necesario realizar más estudios que desvelen las complejas interacciones
que se establecen en ellos (Martínez and Pugnaire, 2009).
2. El estrés salino
La salinización de los suelos es uno de los mayores problemas no sólo a nivel ecológico
sino también agronómico. A nivel ecológico es la causante de la pérdida de
comunidades naturales de plantas y acelera los procesos de degradación del suelo y
desertificación (Alguacil et al., 2011). La salinidad es además el estrés abiótico que
afecta más negativamente al crecimiento vegetal y por tanto a la producción agrícola,
además de limitar el uso de nuevas áreas potenciales de cultivo. Las plantas pueden
clasificarse en base a su tolerancia a la salinidad como halófitas (tolerantes) y glicófitas
(sensibles), habiendo grandes diferencias dentro de cada grupo en base al nivel de
tolerancia (Greenway and Munns, 1980). Debido a que la mayor parte de los cultivos
son glicófitos, el exceso de sales en el suelo ocasiona pérdidas importantísimas en las
explotaciones agrícolas (Pitman and Läuchli, 2002). Los factores ambientales están
estrechamente relacionados con la distribución de las zonas afectadas por salinidad. Por
tanto en los climas áridos y semiáridos, caracterizados por escasez de lluvias,
temperaturas extremas y alta velocidad de evaporación (Brito et al., 2011), el principal
factor limitante de la fertilidad de los suelos y la productividad de los cultivos lo
constituye la salinidad (Evelin et al., 2009). A nivel mundial hay más de 800 millones
de hectáreas afectadas por la salinidad, más del 6% del área total de la superficie
terrestre (Munns and Tester, 2008). En particular, en la cuenca mediterránea alrededor
de 16 millones de hectáreas están afectadas por la salinidad y en la España la cifra
asciende hasta 840.000 hectáreas (Serrano, 2009). Además de las causas naturales, la
actividad humana, en concreto las malas prácticas de cultivo y el riego, han contribuido
al incremento alarmante de sales en el suelo (Mahajan and Tuteja, 2005).
17
Introducción
• Estrés osmótico: la salinidad del suelo dificulta la extracción de agua por parte
de las raíces ya que son expuestas a un bajo potencial osmótico. Esta
disminución de la capacidad de las plantas de absorber agua del suelo supone
una reducción de la expansión foliar y una pérdida de turgencia, siendo más
evidente la reducción del crecimiento en la parte aérea que en las raíces (Munns,
2002). Por tanto las plantas son expuestas a sequía fisiológica y tienen que
mantener bajo el potencial osmótico interno ya que a altas concentraciones de
Na+ en el medio extracelular las plantas deben evitar que el agua salga de las
raíces al suelo (Ruiz-Lozano et al., 2012). Para mantener el potencial osmótico
interno y la actividad citosólica el agua ha de pasar de la vacuola al citosol. Esto
conlleva una reducción en el turgor, en la expansión celular, en la velocidad de
división celular y afecta al desarrollo reproductivo (Munns and Tester, 2008).
18
Introducción
19
Introducción
Debido al efecto negativo que produce el estrés salino en las plantas, éstas han
desarrollado mecanismos bioquímicos y moleculares para paliar el efecto negativo de la
salinidad (Figura 2). Para la mayoría de las plantas, el mecanismo para conferir mayor
tolerancia a la salinidad es restringir la absorción de Na+ y Cl- y mantener la del resto de
macronutrientes a niveles normales (Teakle and Tyerman, 2010).
20
Introducción
3. Las Micorrizas
La mayoría de las plantas que crecen sobre la corteza terrestre viven asociadas, en
forma de simbiosis mutualista, con ciertos hongos del suelo, constituyendo las llamadas
micorrizas, término que deriva del griego mykos (hongo) y riza (raíz). Este término fue
utilizado por primera vez por el botánico alemán A.B. Frank a finales del siglo XIX
(Frank, 1885), haciendo referencia a la simbiosis observada entre las raíces de ciertos
árboles con un micelio fúngico a la que ya, en aquel momento, supuso la función de
21
Introducción
Sin embargo, no fue hasta los años 50 cuando se comenzó a poner de manifiesto
la importancia real y el significado de estas asociaciones, así como su presencia en la
práctica totalidad de los sistemas suelo-planta (Barea and Jeffries, 1995). El hongo,
habitante común de los suelos, contacta las raíces y coloniza biotróficamente la corteza
de las mismas, sin causar daño a la planta y sin llegar a activar por completo su reacción
de defensa (Smith and Read, 2008). Una vez que el hongo ha colonizado la raíz,
desarrolla un micelio externo que coloniza el suelo que rodea la raíz y ayuda a la planta
a adquirir nutrientes minerales y agua, además de conferirle una mayor resistencia a los
estreses ambientales (Barea et al., 2012). Según esto, en la mayoría de los casos, el
órgano de captación de nutrientes de la planta sería la micorriza y no la raíz
propiamente dicha (Harley and Smith, 1983). A su vez la planta hospedadora
proporciona al hongo simbionte (heterótrofo) compuestos carbonados procedentes de la
fotosíntesis, así como un nicho ecológico protegido donde poder completar su ciclo de
vida (Brundrett, 2004).
22
Introducción
Las bases fundamentales sobre las que se establece la simbiosis son nutritivas:
los hongos MA reciben fotosintatos de la planta a cambio de una mejora en la toma de
nutrientes, fundamentalmente fósforo, que es el elemento limitante en el ecosistema
terrestre (Bucher, 2007), pero también potasio, calcio, magnesio, azufre, hierro, zinc,
cobre, manganeso (Boomsma and Vyn, 2008), así como agua del suelo, dada la mayor
accesibilidad del micelio externo del hongo a recursos del suelo más distantes del
sistema radical (Ferrol and Pérez-Tienda, 2009). No obstante, la asociación genera otros
beneficios, entre los que destacan una mayor resistencia de la planta micorrizada al
ataque de patógenos del sistema radical (Hooker et al., 1994; Pozo et al., 2009), a la
presencia de metales pesados en el suelo (del Val et al., 1999; González-Guerrero et al.,
2009), al estrés hídrico (Augé, 2001; Ruíz-Lozano and Aroca, 2010) o a condiciones
extremas de pH del suelo (Clark et al., 1999; Oliveira et al., 2005; Cornejo et al., 2008).
Además, la red de hifas extrarradicales y ciertas sustancias secretadas por el hongo
durante la asociación favorecen la formación de agregados estables en el suelo y, por
tanto, la conservación de la estructura física del mismo (Wright and Upadhyaya, 1998;
Jeffries and Barea, 2012). Es por esto que las MA desempeñan un papel fundamental en
la supervivencia y desarrollo de las plantas, sobre todo, en suelos sometidos a
condiciones de estrés (sequía, salinidad, cambios bruscos de temperatura, deficiencia de
nutrientes), como los que caracterizan a los ecosistemas mediterráneos (Requena et al.,
2001; Sánchez-Castro et al., 2012).
23
Introducción
Los primeros intentos de clasificar los hongos MA datan de finales del siglo XIX y
comienzos del XX y se basaron exclusivamente en criterios morfológicos referenciados
en las esporas. En estos estudios estos hongos se incluyeron en la familia Endogonaceae
dentro del phylum Zygomycota (Gerdemann and Trappe, 1974). En los últimos años la
sistemática del grupo de hongos formadores de MA ha sufrido varias modificaciones,
sobre todo por la incorporación de técnicas moleculares en el estudio de la filogenia de
estos hongos. Actualmente se ha demostrado la naturaleza monofilética de este grupo de
hongos mediante el análisis de 18S ADNr. Esto ha permitido incluirlos en un nuevo
phylum, denominado Glomeromycota (Schüβler et al., 2001). En la actualidad este
phylum es motivo de debate y posee dos posibles clasificaciones que se muestran a
continuación (Tablas 1 y 2).
24
Introducción
25
Introducción
Apresorio
Esporas
Extrarradical
Intrarradical BAS-espora
Figura 3. Ciclo de vida de los hongos formadores de MA (Tomado de Porcel et al. 2012).
Las MA tienen dos componentes bien diferenciados: la “fase extrarradical” del hongo
en la que se incluyen el micelio externo, esporas y ocasionalmente células auxiliares, y
la “fase intrarradical” que incluye hifas intra e intercelulares, arbúsculos y, en algunas
especies, vesículas (Figura 4). La colonización de la raíz sólo ocurre en la epidermis y el
parénquima cortical, ya que el hongo nunca llega a penetrar en el cilindro vascular ni las
zonas meristemáticas (Barea et al., 2008).
26
Introducción
Figura 4. Esquema de las estructuras anatómicas de las MA. Abreviaturas: A, arbúsculo; AP,apresorio;
E, espora; H, hifa intercelular; M, micelio extraradical; O, ovillo; V, vesícula. (Tomado de Palenzuela
and Barea, 2002).
El proceso de formación de las MA tiene lugar tras una sucesión de interacciones entre
el hongo y la planta, que van a dar lugar a una integración morfológica y funcional de
ambos simbiontes (Gianinazzi-Pearson et al., 2004). Como resultado, la planta acepta la
colonización por parte del hongo sin mostrar una reacción de defensa persistente
(Dumas-Gaudot et al., 2000; Pozo et al., 2002). El establecimiento de la simbiosis es el
resultado de un continuo diálogo molecular y un esfuerzo mutuo para aceptar el
intercambio de señales de reconocimiento tanto de la planta como del hongo (Gollotte et
al., 2002; Vierheilig and Piché, 2002). La identificación y clonación de los genes de la
planta implicados en el programa simbiótico en MA (Gianinazzi-Pearson and
Brechenmacher, 2004) y el conocimiento de los programas de señales involucrados en
la formación y funcionamiento de la simbiosis (Harrison, 2005) son objeto de gran
interés en la actualidad.
27
Introducción
28
Introducción
beneficia la diversidad y sucesión de las plantas (van der Heijden et al., 1998; Hart and
Klironomos, 2002).
29
Introducción
Todos estos estudios han sugerido varios mecanismos para explicar la mejora en la
tolerancia de las plantas al estrés salino:
Sin embargo los mecanismos que permiten a las plantas colonizadas por MA
tener mayor tolerancia a la salinidad están todavía lejos de ser comprendidos en su
totalidad. El mayor problema para resolver los mecanismos implicados en la mejora en
la tolerancia al la salinidad, es que la tolerancia salina es un mecanismo complejo y
codificado por familias de genes, lo que implica a varios mecanismos tanto fisiológicos
como bioquímicos y que además varían entre especies (Mian et al., 2011). Por lo tanto
los estudios del impacto de la colonización MA en la tolerancia de las plantas al estrés
salino se encuentran con este problema, entre otros. Además se debe prestar especial
atención a los aislados de MA en ambientes salinos, ya que deberían de estar fisiológica
30
Introducción
31
Introducción
6. Bibliografía
32
Introducción
33
Introducción
34
Introducción
35
Introducción
36
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37
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38
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39
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40
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Tester M, Davenport R. 2003. Na+ tolerance and Na+ transport in higher plants.
Annals of Botany 91, 503-527.
Therond P, Bonnefont-Rousselot D, Davit-Spraul A, Conti M, Legrand A. 2000.
Biomarkers of oxidative stress: an analitical approach. Current Opinion in
Clinical Nutrition and Metabolic Care 3, 373-384.
Thompson JD, Lavergne S, Affre L, Gaudeul M, Debussche M. 2005. Ecological
differentiation of Mediterranean endemic plants. Taxon 54, 967-976.
Tian CY, Feng G, Li XL, Zhang FS. 2004. Different effects of arbuscular mycorrhizal
fungal isolates from saline or non-saline soil on salinity tolerance of plants.
Applied Soil Ecology 26, 143-148.
Tuteja N. 2007. Mechanisms of high salinity tolerance in plants. Methods in
Enzymology 428, 419-438.
van der Heijden MGA, Bardgett RD, van Straalen NM. 2008. The unseen majority:
soil microbes as drivers of plant diversity and productivity in terrestrial
ecosystems. Ecology Letters 11, 296-310.
van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R,
Boller T, Wiemken A, Sanders IR. 1998. Mycorrhizal fungal diversity
determines plant biodiversity, ecosystem variability and productivity. Nature
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Vierheilig H, Piché Y. 2002. Signalling in arbuscular mycorrhiza: Facts and
hypotheses. Flavonoids in Cell Function 505, 23-39.
Wheeler JH, Kostbade JT. 1990. World Regional Geography: Saunders College
Publishing.
White PJ, Broadley MR. 2001. Chloride in soils and its uptake and movement within
the plant: a review. Annals of Botany 88, 967-988.
Wilde P, Manal A, Stodden M, Sieverding E, Hildebrandt U, Bothe H. 2009.
Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt
marshes. Environmental Microbiology 11, 1548-1561.
Wright SF, Upadhyaya A. 1998. A survey of soils for aggregate stability and
glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi.
Plant and Soil 198, 97-107.
Wu QS, Zou YN, He XH. 2010. Contributions of arbuscular mycorrhizal fungi to
growth, photosynthesis, root morphology and ionic balance of citrus seedlings
under salt stress. Acta Physiologiae Plantarum 32, 297-304.
Xu G, Magen H, Tarchitzky J, Kafkafi U. 2000. Advances in chloride nutrition.
Advances in Agronomy 68, 96-150.
Yaalon DH. 1997. Soils in the Mediterranean region: What makes them different?
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Yamato M, Ikeda S, Iwase K. 2008. Community of arbuscular mycorrhizal fungi in a
coastal vegetation on Okinawa island and effect of the isolated fungi on growth
of sorghum under salt-treated conditions. Mycorrhiza 18, 241-249.
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Zandavalli RB, Dillenburg LR, de Souza PVD. 2004. Growth responses of Araucaria
angustifolia (Araucariaceae) to inoculation with the mycorrhizal fungus Glomus
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42
CAPÍTULO 1
CHAPTER 1
33
34
Capítulo 1
Resumen
45
46
Chapter 1
Beatriz Estradaa, Maria Beltrán-Hermosob, Javier Palenzuelaa, Koji Iwasec, Juan Manuel
Ruiz-Lozanoa, José-Miguel Bareaa* and Fritz Oehld*
a
Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del
Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.
b
Departamento de Ingeniería Civil, Edificio Politécnico, Universidad de Granada, Campus de
Fuentenueva, 18071, Spain.
c
Department of Natural and Environmental Science, Teikyo University of Science,
2525 Yatsusawa, Uenohara 409-0193, Japan.
d
Agroscope Reckenhoz-Tänikon Research Station (ART), Ecological Farming Systems,
Reckenholzstrasse 191, CH-8046, Zürich, Switzerland.
47
Chapter 1
Abstract
The use of AM fungi adapted to salinity could be a critical issue for success in
recovering saline areas either in natural environments or in agricultural lands that
became salinized due to a non appropriate land use or under a climate change scenario.
Therefore looking for salinity-adapted AM fungi is fundamental to explore these
possibilities. Despite its important role, there is little information on the distribution and
abundance of the different mycorrhizal associations in saline environments in European
Mediterranean areas. In the present study the community of AM fungi is investigated in
the rhizosphere of a representative plant species adapted to saline Mediterranean areas,
Asteriscus maritimus (L.). Samples of the rhizosphere of twenty A. maritimus plants
were taken in two different areas of the Cabo de Gata Natural Park: a saline dune and a
salt marsh. A total of 30 spore morphotypes belonging to three classes, five orders, nine
families and 13 genera were found in the dune and salt marsh. In the rhizosphere of the
salt marsh spore densities were six times higher than in the dunes, although the diversity
was quite similar in both systems. One new AM fungal species has been described in
the dune and recently published, and four species could not been unequivocally
identified, suggesting possible new species that still need some further analysis before
publication. These results should be taken into account when designing effective
inocula for revegetation programmes.
1. Introduction
48
Chapter 1
In these areas, where conditions are unfavourable for plant growth, arbuscular
mycorrhizal (AM) fungi play an essential role (Trappe 1981). AM fungi are ubiquitous
soil inhabitants belonging to the phylum Glomeromycota which establish mutualistic
symbiotic associations with most land plants (Smith and Read 2008). The symbiosis
between plant and AM fungi is one of the plant strategies for growing under a variety of
stress conditions (Entry et al. 2002). The ecological impact of AM fungi is particularly
relevant for arid and semi-arid ecosystems where they would enable greater plant
tolerance of environmental stresses characteristic of these ecosystems (Requena et al.
1996; Allen 2007).
It is well documented that AM fungi improve plant growth and health: they facilitate
nutrient uptake (van der Heijden et al. 2006), confer drought and salt tolerance (Ruíz-
Lozano and Azcón 2000; Cantrell and Linderman 2001; Marulanda et al. 2009), protect
plants against pathogens (Slezack et al. 2000; Pozo et al. 2009), have positive impacts
on soil aggregate stability and water infiltration (Rillig and Mummey 2006), and
prevent soil erosion (O'Dea 2007). Because of the key ecological functions played in
soil and plants by AM symbiosis, a diverse community of AM fungi is necessary for the
development and maintenance of plant diversity (Jeffries and Barea 2001), contributing
to plant community productivity in different ecosystems (van der Heijden et al. 1998).
Therefore, as AM fungi play important roles in the vigour of plant communities and the
restoration of disturbed ecosystems (Renker et al. 2004), assessment of the native
species composition is an important issue in several contexts and the basis to produce
AM inoculum for selected plant species to be used in the revegetation processes (Ferrol
et al. 2004). Moreover, the use of AM fungi adapted to salinity could be a critical issue
for success in recovering saline areas either in natural environments or in agricultural
lands that became salinized due to inappropriate land use. Therefore looking for
salinity-adapted AM fungi is fundamental to explore these possibilities (Porcel et al.,
2012; Estrada et al., 2012). Despite its important role, there is little information on the
distribution and abundance of the different mycorrhizal associations in saline
environments in European Mediterranean areas.
The aim of the present work was to analyse the diversity of AM fungal species in
sensitive ecosystems affected by desertification and salinity in Southeast Spain,
selecting two ecosystems: salt marshes and dunes. Cabo de Gata Natural Park is the
most arid ecosystem in Europe (Geiger 1973) and was selected as the target area. AM
fungi were screened in the rhizosphere of Asteriscus maritimus (L.), an halophyte
member of the Asteraceae family, highly mycotrophic (Schaede 1962), and native of
lands surrounding the Mediterranean Sea, especially Spain (Lendínez et al. 2011).
Moreover, as the plant spreads its stems over the soil they protect the soil from erosion
(Alcaraz et al. 1997). It is found both in salt marshes and dunes. However, differences
in the AM fungal communities associated with this plant can be assumed dependent on
the ecosystems. For this purpose AM fungi were studied either directly in samples from
49
Chapter 1
the target soil or from one year bait plant cultures established to increase AM fungal
spore population, to reveal their maximum complement possible.
To identify the different AM fungal species in natural saline sites, morphological and
developmental criteria were applied. The morphological identification of AMF can
accurately be done with spores (Oehl et al. 2011), while the fungal structures within the
roots (intraradical hyphae, arbuscules, vesicles,) may vary only within the family or
order but not at the species level (Dodd et al. 2000). The molecular characterization of
AMF in roots is faced with the problem that only about 5% of the total DNA of a root
belongs to the fungi (Toth et al. 1991). Our primary purposes were to understand the
species composition of AM fungi in the rhizosphere of A. maritimus, and to elucidate
the distribution patterns of AM fungi communities related to habitats.
The study was carried out in a representative saline Mediterranean ecosystem in the
Natural Park Cabo de Gata in Almería (Andalucía, Spain). The climate is dry and hot,
with an average annual temperature of 18.5ºC, irregular rainfall occurring mostly in
autumn with a mean annual rainfall less than 200mm and an annual solar hours of
2.960. Two habitats were selected: a natural sand dune (36º44´41´´N 02º07´26´´W) and
a salt marsh (36º45´24´´N 02º13´17´´W).
50
Chapter 1
For the analyses of AM fungal spores, 50 g of each rhizospheric soil were taken in
March 2010, and 25 g in March 2011 from each trap cultures. AM fungal spores, both
from the field collected soil and from the trap cultures, were isolated, identified and
counted separately for each location. They were separated from the soil samples by a
wet sieving and decanting method, followed by sucrose centrifugation process
(Sieverding 1991). After centrifugation, the supernatant was poured through a 50 μm
mesh and quickly rinsed with tap water. Turgid spores (suggesting viability) were
counted under a dissecting microscope using up to 90-fold magnification. For
identification of AMF species, spores (about 50-70% of the total numbers) were picked
under the dissecting microscope and mounted in polyvinyl alcohol-lactic acid-glycerine
(PVLG) (Koske and Tessier 1983) or PVLG mixed 1:1 (v/v) with Melzer's reagent
(Brundrett et al. 1994) for permanent slides. The spores were then examined using a
compound microscope at up to 400-fold magnification. The AMF species identification
was based on current species descriptions and identification manuals (e.g. Schenck and
Pérez 1990); International Culture Collection of (Vesicular) Arbuscular Mycorrhizal
Fungi (http://invam.caf.wvu.edu/fungi/taxonomy/speciesID.htm); Department of Plant
Pathology University of Agriculture in Szczecin, Poland
(http://www.agro.ar.szczecin.pl/~jblaszkowski/)), and on own type specimen analyses
of almost all AMF species described. In this study, an AMF “species” is either a clearly
identified species based on its spore morphology, or a species as yet unknown to us. We
follow the glomeromycotean classification of Oehl et al. (2011) recently published in
the new journal of the International Mycological Association on request. Photographs
were taken with a Leica DFC 290 digital camera on a Leitz Laborlux S compound
microscope using Leica Application Suite Version V 2.5.0 R1 software.
51
Chapter 1
The species richness is defined as the total number of species found in a community.
The specific density (Di) is the proportion of individuals of one particular species in the
sample (i) relative to the total number of individuals (N).
Di = ni / N
In the present work species richness and specific density have been evaluated at the
dune and salt marsh.
3. Results
All surveyed samples were colonized by AMF and formed typical arbuscular
mycorrhizal structures. Intra- and intercellular hyphae, vesicles and arbuscules were
abundant in the root tissues.
At the dune site, the soil was alkaline with pH 8.2, organic carbon 15.3 g kg-1, total
nitrogen 1.9 g kg-1, available P 27.0 mg kg-1, and soil electrical conductivity 0.5 dS m-1.
At the salt marsh, the soil was alkaline with pH 8.7, organic carbon 2.6 g kg-1, total
nitrogen 0.3 g kg-1, available P 47.0 mg kg-1 and soil electrical conductivity 3.95 dS m-1.
The AMF spore densities differed among the sites. Salt marsh had regularly higher
spore densities than the dune, both at the rhizosphere soil and in the bait cultures. In
March 2010, the spore densities were 1.2 and 7.2 g-1 soil at the dune and the salt marsh,
respectively. After one year in the trap cultures, the spores densities were more than
doubled, with 2.8 and 17.8 spores at the dune and the salt marsh. The relation between
dune and salt marsh remained the same, being the spore density around six times higher
at the salt marsh than at the dune.
In total 30 AMF species could be distinguished from the field samples across all sites
and both years of survey. They belong to all three current classes, to all five current
orders, to nine families and thirteen genera currently known in the Glomeromycota
(Table 1). The majority of the isolated species (16) were of the order Glomerales (3
52
Chapter 1
In the field samples of March 2010, 16 AMF species belonging to 9 genera were found
in the sand dune. After one year of bait culturing, 15 AMF species reproduced spores in
the cultures belonging to 10 genera. From field and bait cultures, a total of 23 AMF
species were detected belonging to 11 AMF genera were identified. Of these species, 7
were found recovered only from the field samples and 8 only from the bait cultures,
while 9 species were detected both in the field samples and the bait cultures (Table 1).
In 2010, the species detected (in order of decreasing specific density), were: Se.
constrictum, Diversispora sp. BEV4, Cl. claroideum, Fu. coronatus, Gl. badium, Gl.
intraradices, Gl. macrocarpum, Gl. sp. BEV2, Sc. calospora, Gl. rubiforme, Ra.
persica, Ac. scrobiculata. After one year of bait culturing, the species recovered from
the sand dune (in order of decreasing specific density, see Figure 1) were: Se.
constrictum, Fu. coronatus, Cl. claroideum, Pa. dominikii, Gl. intraradices, Cl.
etunicatum, Di. clara, Di. versiformis, Fu. mosseae, Gl. microaggregatum, Sc.
calospora, Di. aurantia, Par. occultum, Ar. trappei, Pa. franciscana, Ac. scrobiculata.
In the field and in the bait cultures, Se. constrictum was the most abundant species.
In the salt marsh system, a total of 24 AMF species, also belonging to 11 AMF genera,
were detected (Table 1). In the field samples from the salt marsh, 20 morphospecies
were identified in 2010 belonging to 10 AMF genera, and after one year of bait
culturing, 12 AMF species had reproduced spores belonging to 7 genera. Of these
species, 11 were found identified only from the field samples and 4 only from the bait
cultures, while 9 species were recovered from both the field samples and the bait
cultures.
In 2010, the species detected (in order of decreasing specific density, see Figure 1),
were: Se. constrictum, Diversispora sp. BEV4, Gl. sp. BEV2, Entrophospora sp. BEV3,
Gl. intraradices, Cl. claroideum, Fu. coronatus, Sc. calospora, Gl. macrocarpum, Fu.
mosseae, Fu. geosporus, Gl. badium, Cl. etunicatum, Ra. persica. After one year of bait
53
Chapter 1
culturing, the species recovered from the sand dune (in order of decreasing specific
density, see Figure 1) were: Par. occultum, Gl. intraradices, Fu. coronatus, Cl.
claroideum, Cl. etunicatum, Se. constrictum, Fu. mosseae, Di. versiformis, Gl.
microaggregatum, Di. aurantia, Fu. geosporus, Ar. myriocarpa, Ar. trappei. In the field
samples, Se. constrictum was the most abundant species, but not in the trap cultures
Par. occultum had the highest specific density.
Fu. coronatus
Fu. geosporus
Fu. mosseae
1
Gl. badium
Gl. intraradices
0.9 Gl. macrocarpum
Gl. sp. BEV1
Gl. magnicaule
0.8
Gl. sp. BEV2
Specific AMF density (Di)
Gl. microaggregatum
0.7 Gl. microcarpum
Gl. rubiforme
0.6 Se. constrictum
Cl. claroideum
Cl. etunicatum
0.5 En. sp BEV3
Di. aurantia
0.4 Di. clara
Di. versiformis
Di. sp. BEV4
0.3
Ac. scrobiculata
Pa. dominiki
0.2 Pa. franciscana
Ra. persica
Ra. verrucosa
0.1
Sc. calospora
Am. gerdemannii
0 Ar. myriocarpa
Dune 2010 Dune 2011 S. marsh 2010 S. marsh 2011 Ar. trappei
Par. occultum
Figure 1. Specific density (Di) of AMF found in dune and salt marsh at two consecutive years 2010 and
2011 (field-collected soil and bait cultures respectively).
AMF species and genus richness was similar in both ecosystems (Table 1) with 23
respective 24 out of 30 AMF species and each 11 out of 13 AMF genera detected,
respectively. Ambispora and Entrophospora were the two genera found in the study
area that were not detected in the dune systems but only in the salt marsh system. On
the other hand, Acaulospora and Pacispora species were not found in the salt marsh
while they had been revealed from the dune (Table 1). Other remarkable difference
between both scenarios was the presence of Fu. geosporus and Entrophospora sp.
BEV3 only in the salt marshes. Also Gl. microcarpum, Gl. sp. BEV1, Ra. verrucosa,
Am. gerdemanni and Ar. myriocarpa were only revealed from the salt marshes, while,
beside Ac. scrobiculata, Pa. dominikii and Pa. franciscana, also Gl. magnicaule and Gl.
rubiforme were only detected in the dune but not in the salt marsh.
54
Chapter 1
Table 1. Presence and species richness of AMF at dune and salt marsh directly from field-collected soil
samples in 2010 and from trap cultures in 2011.
Sand Dune Salt Marsh
Trap Trap
In field culture In field culture
AMF species 2010 2011 2010 2011
GLOMERALES
Glomeraceae
Funneliformis coronatus x x x x
Fu. geosporus x x
Fu. mosseae x x x
Glomus badium x x
Gl. intraradices x x x x
Gl. macrocarpum x x
Gl. sp. BEV1 x
Gl. magnicaule x
Gl. sp. BEV2 x x
Gl. microaggregatum x x x x
Gl. microcarpum x
Gl. rubiforme x
Septoglomus constrictum x x x x
Entrophosporaceae
Claroideoglomus claroideum x x x x
Cl. etunicatum x x x
Entrophospora sp. BEV3 x
DIVERSISPORALES
Diversisporaceae
Diversispora aurantia x x
Di. clara x
Di. versiformis x x
Di. sp. BEV4 x x
Acaulosporaceae
Acaulospora scrobiculata x x
Pacisporaceae
Pacispora dominiki x x
Pa. franciscana x x
GIGASPORALES
Racocetraceae
Racocetra persica x x
Ra. verrucosa x
Scutellosporaceae
Scutellospora calospora x x x
ARCHAEOSPORALES
Archaeosporaceae
Ambispora gerdemannii x
Archaeospora myriocarpa x x
Ar. trappei x x
PARAGLOMERALES
Paraglomeraceae
Paraglomus occultum x x
-1 -1
AMF species richness site year 16 16 20 23
Total AMF species richness site-1 23 24
AMF species richness of study 30
55
Chapter 1
4. Discussion
56
Chapter 1
However, morphotypes assigned to species such as Fu. geosporus, Gl. sp. BEV1, Gl.
microcarpum, Entrophospora sp. BEV3, Ra. verrucosa, Ambispora gerdemannii and
Archaeospora myriocarpa were only found in plants from the salt marsh, while Gl.
magnicaule, Gl. rubiforme, Di. clara, Acaulospora scrobiculata, Pacispora dominiki
and, Pa. franciscana were only detected in the rhizosphere of A. maritimus grown in the
dune.
Since the plant species is not an experimental variable, the reasons for similarities and
differences of the presence of morphotypes should be based on the differential
characteristics between both areas. Specifically, the levels of pH, available phosphorus,
and salinity, have been described as factors that influence the distribution of AM fungi
(Johnson-Green et al. 2001; García and Mendoza 2008).
Similar reasons could also explain the higher density of AMF spores found in the soil
sampled in the A. maritimus rhizosphere of the salt marsh compared to the rhizospheric
samples of the dune at both years of sampling. Another criterion of variability between
the dune and the salt marsh is the spore density of specific species that occurs in some
cases.
Regarding the similarities and discrepancies at the AMF species found in this study,
relevant facts deserve discussion. For instance, it is noteworthy that fungi belonging to
the family Glomeraceae were the predominant sporulators in field and bait conditions
and also presented the highest number of diversity of morphotypes. These species may
be more adapted in adjusting patterns of sporulation to environmental stress conditions
(Jacobson 1997). A similar restriction in species composition to mostly fungi belonging
to Glomus spp. was previously reported in arid and semiarid ecosystems (Jacobson
1997; Stutz et al. 2000).
Previous work indicated that saline soils contain up to 80% of all spores belonging to
one single AMF species, Glomus geosporum (= Fu. geosporus; Carvalho et al. 2001;
Hildebrandt et al. 2001; Landwehr et al. 2002; Grzybowska 2004; Sonjak et al. 2009).
In agreement with the bibliography, Fu. geosporus has been found in the salt marsh,
both at field site and trap culture. However, we found a higher AMF species diversity
than those published and Fu. geosporus was not the most abundant species. In the field
samples, Se. constrictum was the most abundant species in both habitats while in the
bait cultures it only remained predominant in the dune but in the salt marsh Par.
occultum took its place. Both fungi have been widely described in arid and saline
environments: Se. constrictum (Requena et al. 1996; Beena et al. 2001; Kowalchuk et
al. 2002; Ferrol et al. 2004; Rodríguez-Echeverría and Freitas 2006; Wilde et al. 2009;
Hammer et al. 2011), and the genus Paraglomus (Ferrol et al. 2004; Tapia-Goné et al.
2008; Alguacil et al. 2011).
Some Glomeraceae species, Gl. intraradices, Fu. mosseae and Cl. etunicatum for
instance, have been found in saline environments (Aliasgharzadeh et al. 2001;
Landwehr et al. 2002; Tapia-Goné et al. 2008; Sonjak et al. 2009; Wilde et al. 2009;
Camprubí et al. 2010; Yang et al. 2010; Hammer et al. 2011). In the Glomerales order,
57
Chapter 1
three species could not been unequivocally identified, suggesting possible new species;
two of them belonging to the Glomeraceae family, Gl. sp. BEV1 and Gl. sp. BEV2, and
the other to the Entrophosporaceae family, Entrophospora sp. BEV3. The order
Diversisporales gathers the other unidentified species, Diversispora sp. BEV4, and a
new species that have been recently published, Di. clara (Estrada et al. 2011). For
further identification studies, bait cultures have been established to propagate those
unknown fungi.
In this study, only one Acaulosporaceae, Ac. scrobiculata, could be found in the dune,
where the genus Pacispora was very common and widely represented but not in the salt
marsh. Controversially Wilde et al. (2009) found a species of the genus Pacispora, Pa.
scintillans, in the rhizosphere of Puccinelia maritima in Terschelling salt marsh.
Our results also report the occurrence of Racocetra persica among other Gigasporales
morphotypes. This species has been recovered in the Northwestern coast of Italy
(Turrini et al. 2008) and had previously been described from other sand dunes (Koske
and Walker 1985; Blaszkowski and Tadych 1997; Rodríguez-Echeverría and Freitas
2006; Camprubí et al. 2010). Contrary to some results, we did not find any Gigaspora
species in our samples while other authors found several morphotypes (Selvaraj and
Kim 2004; Rodríguez-Echeverría and Freitas 2006; Tapia-Goné et al. 2008). The latter
may be explained according to Turrini et al. (2008) who pointed out that Gigaspora
species are rare in ecosystems with anthropogenic disturbance.
All the information from this study should be considered when designing the AM fungal
inoculum composition for revegetation programs both in natural and agricultural lands
that have become salinized. Schwartz et al. (2006) discussed the use of appropriate
inoculum in restoration programmes and showed that the application of inappropriate
inoculum could result in the upcoming or survival of invasive plant species and the out-
competition of native fungal strains. Thus, the use of autochthonous fungi should be
favoured. Several studies showed the better benefit of native than non native AMF
isolates, which appear to be physiologically and genetically adapted to the stress
conditions of the target environment, in the inoculation strategies used in revegetation
programs of degraded ecosystems (Herrera et al. 1993; Ferrol et al. 2004; Moora et al.
2004; Renker et al. 2004; Oliveira et al. 2005; Querejeta et al. 2006; Alguacil et al.
2011). In addition to that, it may be advisable to use not only one single AMF species
for restoration practices but a mix of a locally adapted AMF community in order to be
able to support a diverse plant cover (Klironomos 2003; Vogelsang et al. 2006).
This study confirms the existence of a rich diversity of AM fungi in the rhizosphere
soils of Spanish saline Mediterranean ecosystems. As inadequate inocula should be
avoided due to possible negative effects on plant species and the plant community, there
is a need to analyze the fungal community associated with target plant species in its
natural environment. In restoration programmes, indigenous inocula should be favoured
over exotic fungi, as the consequences of introduction of non-native fungi on plant
species and plant community structure cannot be precisely predicted. Therefore, this
58
Chapter 1
5. Conclusions
Acknowledgements
This work was financed by two research projects supported by Junta de Andalucía
(Spain). Projects P06-CVI-01876 and P11-CVI-7107.
59
Chapter 1
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Selvaraj T, Kim H (2004) Ecology of Vesicular-Arbuscular Mycorrhizal (VAM) fungi
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Sieverding E (1991) Vesicular-arbuscular mycorrhiza management in tropical
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65
CAPÍTULO 2
CHAPTER 2
Reprinted from Mycotaxon, 2011, 118: 73-81 (Estrada B, Palenzuela J, Barea J.M,
Ruiz-Lozano J.M, da Silva G.A and Oehl F.)
Capítulo 2
Resumen
En las dunas del Parque Natural Cabo de Gata (Almería, Andalucía, sur de España) se
encontró una nueva especie de Diversispora (Glomeromycetes) en la rizosfera de
Asteriscus maritimus, una especie vegetal especialmente adaptada a ambientes salinos.
La nueva especie de hongo formador de micorrizas arbusculares forma esporas blancas
brillantes que tienen un tamaño de 79-130 × 75-125 micras y tienen una pared que
consta de tres capas. La hifa sustentoria es como la típica de muchas Diversispora spp.,
de pared delgada, hialina y cilíndrica (o rara vez constreñida) y flexible y frágil por
debajo de los septos que separan el contenido de esporas y de hifas. Los septos se
forman regularmente en la base de las esporas o, con menor frecuencia, en la hifa
sustentoria a corta distancia de la base de las esporas. El análisis filogenético del ITS y
parte del gen ribosómico 28S confirman que D. clara forma un clado independiente y
monofilético dentro de Diversispora.
69
70
Chapter 2
1
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación
Experimental del
Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, Spain
2
Departamento de Micologia, CCB, Universidade Federal de Pernambuco,
Av. Prof. Nelson Chaves s/n, Cidade Universitária, 50670-420, Recife, PE, Brazil
3
Federal Research Institute Agroscope Reckenholz-Tänikon ART,
Organic Farming Systems, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland
71
Chapter 2
Abstract
A new species of Diversispora (Glomeromycetes) was found in saline sand dunes of the
Natural Park Cabo de Gata (Almería, Andalucía, Southern Spain) in the rhizosphere of
Asteriscus maritimus, a plant species especially adapted to saline environments. The
new fungal species forms brilliant white spores that are 79-130 × 75-125 μm and have
one wall consisting of three layers. The subtending hyphae are, as typical for many
Diversispora spp., thin-walled, hyaline, and cylindrical (or rarely constricted) and
flexible and fragile below the septa separating the spore and hyphal contents. The septa
form regularly at the spore bases or, less frequently, in subtending hyphae at short
distances from the spore base. Phylogenetic analyses of the ITS and partial 28S
ribosomal gene confirm that D. clara forms a monophyletic, independent clade within
Diversispora.
Introduction
During recent studies on arbuscular mycorrhiza (AM) fungal diversity in sand dune
systems of the Cabo de Gata Natural Park in Almería (Spain), a brilliant-white new
glomeromycotean fungus was recovered from the rhizosphere of Asteriscus maritimus,
a plant species characteristic of saline Mediterranean environments with elevated soil
electrical conductivity. The new fungus formed spores in AM fungal bait cultures
predominantly in the Asteriscus maritimus rhizosphere. The aims of the present study
were to analyze this particular fungus applying combined morphological and molecular
tools and to describe its characteristics.
Soil samples were taken in February 2010 from the rhizosphere of ten Asteriscus
maritimus plants growing in a natural sand dune system of the Natural Park Cabo de
Gata in Almería (Andalucía, Spain). The site is located at 36°44´41´´N 02°07´26´´W.
The samples, air-dried in the laboratory, were used to analyze selected chemical soil
parameters (pH, soil organic carbon, total nitrogen, soil electrical conductivity) and
spore populations The ten plants and the surrounding rhizosphere soil were also
extracted and used as bait cultures for propagation of AM fungal communities
indigenous to the natural sand dune system. At the site, the soil was sandy and with pH
72
Chapter 2
(H2O) 8.2, organic carbon 15.3 g kg-1, total nitrogen 1.9 g kg-1, available P 27.0 mg kg-1,
and soil electrical conductivity 0.5 dS m-1.
Bait cultures were established and maintained as described in Palenzuela et al. (2008,
2011) by transplanting 10 Asteriscus maritimus plants with their rhizosphere soils into 1
L pots and transferring them to the greenhouse at EEZ in Granada immediately after
sampling. The pots were irrigated three times per week and fertilized every four weeks
with Long-Aston nutrient solution (Hewitt 1966). Pure cultures of the new fungus were
initiated in a mixed-culture of three Allium porrum L. and three Hieracium pilosella L.
plantlets in 750 mL pots grown together at three locations in the pots. The plant
rhizosphere was inoculated with 20 spores per pot and pot location. A sterile mixture of
Terragreen (American aluminum oxide, Oil Dry US special, type III R; Lobbe
Umwelttechnik Iserlohn, Germany) and Loess (mixture 3:1; with pH-KCl 6.2; organic
carbon 0.3%; available P (Na-acetate) 2.6 mg kg-1; available K (Na-acetate) 350 mg kg-1
was chosen as culture substrate (Oehl et al. 2002). So far, pure cultures of the new
fungus have not been obtained.
Morphological analyses
AM fungal spores were separated from the soil samples by a wet sieving process
(Sieverding 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic
acidglycerol (PVLG; Koske and Tessier 1983); a mixture of PVLG and Melzer’s
reagent (Brundrett et al. 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent;
and water (Spain 1990). The spore structure terminology follows Oehl et al. (2003,
2005, 2011a) for species with glomoid or diversisporoid spore formation. Photographs
(Figs. 1-10) were taken with a Leica DFC 290 digital camera on a Leitz Laborlux S
compound microscope using Leica Application Suite Version V 2.5.0 R1 software.
Specimens mounted in PVLG and the PVLG+Melzer’s mixtures were deposited at the
herbaria Z+ZT (ETH Zurich, Switzerland), GDA-GDAC (University of Granada,
Spain), and URM (Federal University of Pernambuco, Recife).
Molecular analyses
Five spores isolated from the trap cultures were surface-sterilized with chloramine T
(2%) and streptomycin (0.02%) (Mosse 1962) and crushed with a sterile disposable
micropestle in 40 μL milli-Q water (Ferrol et al. 2004). PCRs of the crude extracts were
obtained in an automated thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer,
Foster City, California) with a pureTaq Ready-To-Go PCR Bead (Amersham
73
Chapter 2
Results
Diversispora clara Oehl, B. Estrada, G.A. Silva & Palenz., sp. nov. Figs 1-10
MycoBank MB 561583
Sporae albae, 79–130 × 75–125 μm, tunica tribus stratis, 4.3–8.4 μm. Stratum
exterior hyalinum; stratum medium, album ad rarum ochreo-album, 2.8–5.4 μm
crassum; stratum interius album. Hypha adhaerenta, 8–13 μm in diametrum.
Tunica hyphae 1.0–1.8 μm crassa. Strata medium interiusque septo porum
occludentes. Strata exterior mediumque flava colorantes Melzeri.
TYPE: Spain. Andalucía, Almería, Cabo de Gata Natural Park, sand dune, from
the rhizosphere of Asteriscus maritimus (L.) Less. (Asteraceae), isolation date
74
Chapter 2
FIGS 1-10. Diversispora clara: 1-4. Uncrushed spores with a triple-layered spore wall (swl1-3), a single,
cylindrical subtending hypha (sh), and a septum (sp) arising directly or at some distance from the base.
Figs 5–6. Spores crushed to show triple layered spore wall (swl1-3). FIGS 7-8. Spore wall structure in
PVLG + Melzer’s reagent. The swl1 or swl2 (or both) stain light to dark yellow in Melzer’s, while no
such staining reaction is seen on swl3. However, the yellow stain is usually most noticeable in the spore
cell contents. FIG. 9. Septum at spore base formed by swl2 (swl3 is not visible in uncrushed spore). FIG.
10. Septum at spore base formed by swl3.
75
Chapter 2
SPORE WALL 4.3-8.4 μm thick, three-layered (swl1, swl2, swl3; Figs 5-7);
outer layer (swl1) hyaline, 0.8-1.5 μm thick, evanescent and thus often absent in mature
spores; second layer (swl2) bright to (rarely) creamy white, laminated, 2.8-5.4 μm thick
(Fig. 7) in uncrushed spores (sometimes ≤ 6.6 μm in crushed spores when pressure is
applied on the cover slip); inner layer (swl3) brilliant white, 0.7-1.5 μm thick (usually
tightly adherent to swl2 and difficult to observe when < 1.0 μm; see Figs 5-6); both
swl1 and swl2 generally light to dark yellow in Melzer’s reagent (Figs 7-8, with spore
cell contents often a more intense yellow than the surrounding cell wall layers (Fig. 8).
DISTRIBUTION: Known only in the natural sand dune system of the Cabo de
Gata Natural Park in Andalucía in the rhizosphere of Asteriscus maritimus.
MOLECULAR ANALYSES: Phylogenies derived from ITS (Fig. 11) and 28S
(data not shown) rDNA analyses cluster the new fungus within the Diversisporaceae in
a well-separated clade adjacent to several other Diversispora species, Otospora bareae,
and Tricispora nevadensis (Oehl et al. 2011b).
Discussion
76
Chapter 2
however, was not confirmed for D. clara, since the new fungus generally does not form
pigmented spores.
Molecular analyses confirm the species as new: in the phylogenetic tree, D.
clara clusters in a independent, monophyletic clade within a polyphyletic Diversispora.
These sequence analyses also confirm unequivocally using morphology to identify
diversisporoid species within the Glomeromycota (Oehl et al. 2011a,c).
Including D. clara, there are now 14 Diversispora spp. known in the
Glomeromycetes (Oehl et al. 2011a,b). The 9 species that form significantly pigmented,
yellow brown to brown or orange to orange brown spores are D. arenaria (Błaszk. et
al.) Oehl et al., D. aurantia (Błaszk. et al.) C. Walker & A. Schüssler, D. epigaea (B.A.
Daniels & Trappe) C. Walker & A. Schüssler, D. insculpta (Błaszk.) Oehl et al., D.
przelewicensis (Błaszk.) Oehl et al., D. pustulata (Koske et al.) Oehl et al., D. tenera
(P.A. Tandy) Oehl et al., D. trimurales (Koske & Halvorson) C. Walker & A. Schüssler,
and D. versiformis (P. Karst.) Oehl et al. (Oehl et al. 2011a,b). Diversispora celata C.
Walker et al. forms triple-layered, ochre to ivory to pinkish cream spores (Gamper et al.
2009). Only D. spurca (C.M. Pfeiff. et al.) C. Walker & A. Schüssler, D. Eburnea (L.J.
Kenn. et al.) C. Walker & A. Schüssler, and D. gibbosa (Błaszk.) Błaszk. & Kovács
form hyaline to subhyaline spores that are, however, never brilliantwhite as observed for
D. clara. Moreover, D. spurca and D. eburnea have bilayered spore walls (Kennedy et
al. 1999), D. gibbosa has a five-layered wall (Błaszkowski 1997), and D. clara has a
three-layered wall.
In the past, large-spored or sporocarpic AM fungi were described from sand
dune systems, such as Gigaspora, Scutellospora, Pacispora or Glomus species that
were generally easy to isolate and recognize from field samples (Koske & Gemma
1995, Gemma et al. 1989, Błaszkowski 1994). Likewise in the current Cabo de Gata
Natural Park sand dune system, where Funneliformis coronatus (Giovann.) C. Walker
& A. Schüssler, F. mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüssler,
Scutellospora calospora (T.H. Nicolson & Gerd.) C. Walker & F.E. Sanders, Racocetra
persica (Koske & C. Walker) Oehl et al., and Glomus macrocarpum Tul & C. Tul. were
identified (Estrada, unpublished). When sand dune AM fungal communities were
maintained and reproduced in bait cultures, small-spored Glomus spp. were also
sometimes detected (e.g. Błaszkowski et al. 2009a,b, 2010). It was supposed that
species with small, quickly degrading spores were difficult to recover or identify only
from field samples. This might also be true for D. clara, even though its laminated wall
structure is clearly persistent. It will be interesting to see whether future taxonomists
will be able to identify the new species directly from field samples, now that the
existence and morphology of this unique, brilliant-white, conspicuous but small-spored
species is known. Morphological spore and molecular root and spore analyses will
hopefully tell us more about the ecology and biogeography of this fungus that is thus far
known only from a single Asteriscus maritimus rhizosphere in the Natural Park Cabo de
Gata of Almería in southern Spain.
77
Chapter 2
FIG. 11. Diversisporaceae. ITS rDNA-based phylogenetic tree rooted by Acaulospora laevis and A.
lacunosa. Sequences are labeled with database accession numbers. Support values are from neighbor-
joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses. Diversispora
clara sequences are in bold. Only topologies with ≥ 50% bootstrap values are shown. (Consistency Index
= 0.72; Retention Index = 0.84).
78
Chapter 2
Acknowledgments
This study has been supported by the Junta de Andalucía (Spain), project P06-CVI-
01876 and by the Swiss National Science Foundation (SNSF; Project
315230_130764/1). We acknowledge the valuable comments on the manuscript and
revisions of Dr. Bruno Tomio Goto (Universidade Federal do Rio Grande do Norte,
UFRN, Natal, Brazil), Dr. Iván Sánchez-Castro (INRA, Dijon, France) and PD Dr.
Ewald Sieverding (University of Hohenheim, Germany) and appreciate the corrections
by Shaun Pennycook, Nomenclatural Editor, and suggestions by Lorelei L. Norvell,
Editor-in-Chief.
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CAPÍTULO 3
CHAPTER 3
73
74
Capítulo 3
Resumen
Los hongos micorrícicos arbusculares (MA) se ha visto que existen de forma natural en
ambientes salinos y se ha sugerido que las diferencias en el comportamiento de estos
hongos y su eficiencia puede ser debida al origen y la adaptación del hongo MA. Estos
resultados invitan a buscar especies de MA aisladas en ambientes salinos y comparar
sus mecanismos de tolerancia a la salinidad con aquellas especies de áreas no-salinas.
Para ello un aislado de G. intraradices (Gi CdG) perteneciente a una región con graves
problemas de salinidad y afectada por la desertificación se ha comparado con un aislado
de colección de la misma especie, que se usa habitualmente como hongo modelo. Un
experimento in vitro comprobó la capacidad de ambos hongos MA de crecer bajo
niveles crecientes de salinidad en el medio y un experimento in vivo comparó su
eficiencia simbiótica con las plantas de maíz cultivadas bajo condiciones de estrés
salino. El aislado Gi CdG se desarrolló mejor bajo condiciones de salinidad e indujo
considerablemente la expresión de los genes GintBIP, Gint14-3-3 y GintAQP1, si bien
mostró una inducción más baja del gen GintSOD1 que la cepa de colección G.
intraradices. El aislado Gi CdG también estimuló el crecimiento de las plantas de maíz
más que la cepa de colección bajo dos niveles de salinidad. La mayor eficiencia
simbiótica de Gi CdG fue corroborada por el aumento de la eficiencia del fotosistema II
y la conductancia estomática y la menor pérdida de electrolitos mostrada por plantas de
maíz bajo las diferentes condiciones probadas. La mayor tolerancia a la salinidad y la
eficiencia simbiótica exhibida por el aislado Gi CdG en comparación con el G.
intraradices de colección puede ser debido a una adaptación del hongo a los ambientes
salinos. Tal adaptación puede estar relacionada con la importante inducción de la
expresión de los genes que codifican para chaperonas o para acuaporinas del hongo. El
presente estudio muestra que los hongos MA aislados en zonas afectadas por salinidad
pueden ser una herramienta poderosa para mejorar la tolerancia de los cultivos a
condiciones de estrés salino.
85
86
Chapter 3
87
Chapter 3
Abstract
Aims:
Arbuscular mycorrhizal (AM) fungi have been shown to occur naturally in saline
environments and it has been suggested that differences in fungal behaviour and
efficiency can be due to the origin and adaptation of the AM fungus. These findings
invite to look out for AM fungal species isolated in saline environments and compare
their salt-tolerance mechanisms with those of species living in non-saline areas.
Methods:
A fungal strain of G. intraradices (Gi CdG) isolated from a region with serious
problems of salinity and affected by desertification, has been compared with a
collection strain of the same species, used as a model fungus. An in vitro experiment
tested the ability of both AM fungi to grow under increasing salinity and an in vivo
experiment compared their symbiotic efficiency with maize plants grown under salt
stress conditions.
Results:
The isolate Gi CdG developed better under saline conditions and induced considerably
the expression of GintBIP, Gint14-3-3 and GintAQP1 genes, while it showed a lower
induction of GintSOD1 gene than the collection G. intraradices strain. The isolate Gi
CdG also stimulated the growth of maize plants under two levels of salinity more than
the collection strain. The higher symbiotic efficiency of Gi CdG was corroborated by
the enhanced efficiency of photosystem II and stomatal conductance and the lower
electrolyte leakage exhibited by maize plants under the different conditions assayed.
Conclusions:
The higher tolerance to salinity and symbiotic efficiency exhibited by strain Gi CdG as
compared to the collection G. intraradices strain may be due to a fungal adaptation to
saline environments. Such adaptation may be related to the significant up-regulation of
genes encoding chaperones or genes encoding aquaporins. The present study remarks
that AM fungi isolated from areas affected by salinity can be a powerful tool to enhance
the tolerance of crops to saline stress conditions.
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Chapter 3
Introduction
89
Chapter 3
Smith and Read 2008). In the AM symbiosis, plants get nutrients and water resources
that are less available to the plant roots from the fungi, while the fungi receive carbon
compounds from the plant and find a niche to complete their life cycle (Koide and
Mosse 2004). At the same time, AM symbiosis enhances plant tolerance to different
abiotic stresses with osmotic components such as drought and salinity (Augé 2001;
Ruíz-Lozano 2003; Ruíz-Lozano et al. 2006; Jahromi et al. 2008). Although it is clear
that AM fungi mitigate growth reduction caused by salinity, the mechanism involved
remains unresolved (Ruiz-Lozano et al. 2012). Moreover, some studies reveal that
salinity affects directly the fungal development, reducing fungal mycelia formation and
host root colonization (Poss et al. 1985; Juniper and Abbott 2006; Giri et al. 2007;
Sheng et al. 2008). Contrary to those reports, a few other studies reported no reduction
or even increasing fungal development (Hartmond et al. 1987; Aliasgharzadeh et al.
2001; Yamato et al. 2008). This may be related to evolved mechanisms that allow
specific AM fungi to have a higher tolerance to salinity. In fact, mycorrhizal fungi have
been shown by several workers to occur naturally in saline environments (Juniper and
Abbott 1993) and Copeman et al. (1996) suggested that differences in fungal behaviour
and efficiency can be due to the origin of the AM fungus (Ruíz-Lozano and Azcón
2000). These results invite to look out for AM fungal species isolated in saline
environments and compare their salt-tolerance mechanisms with those of species living
in non-saline areas (Porcel et al. 2012).
The main problem to elucidate the mechanisms that allow specific AM fungi to
tolerate salinity is that salt tolerance is a multigenic and complex trait which involves
many physiological and biochemical mechanisms that vary between species (Mian et al.
2011). Thus, when examining putative salt tolerant AM fungi, several aspects involved
in the protection against damage caused by ROS, altered water content or protein
inactivation should be evaluated. In this regard, it is known that to overcome the
oxidative damage generated by stresses such as salinity, all living organisms have
developed antioxidant systems to efficiently scavenge ROS excess. Little is known
about the antioxidant responses in the AM fungi, but studies have demonstrated that
AM fungi possess ROS scavenging systems. These include genes encoding for
superoxide dismutases (SOD) (Lanfranco et al. 2005; González-Guerrero et al. 2010) or
glutaredoxins (GRXs) (Benabdellah et al. 2009), although their involvement in the
fungal responses to salinity have never been studied. On the other hand, aquaporins are
proteinaceous pores present in the membranes of all living organisms that facilitate the
transport of water and other small and neutral solutes (Forrest and Bhave 2007; Maurel
et al. 2008). An aquaporin gene has been described in the AM fungus G. intraradices
which may have a role in the transport of water from mycelium growing under
osmotically favourable conditions to salt-stressed mycelium (Aroca et al. 2009). Finally,
protection mechanisms to prevent protein inactivation by salinity can include the
activity of 14-3-3 proteins or that of chaperone-like proteins such as luminal binding
proteins (BiP). 14-3-3 proteins are binding proteins that regulate the activities of a wide
90
Chapter 3
array of targets via direct protein–protein interactions (Bridges and Moorhead 2004). In
plants, these proteins have a role in regulation of development and stress response
(Chung et al. 1999; Roberts 2003), including salinity (Wang et al. 2002; Xu and Shi
2006). A gene encoding for a 14-3-3 protein has been described in G. intraradices
(Porcel et al. 2006), but its involvement in the fungal response to salinity has not been
investigated so far. Luminal binding proteins (BiPs) are molecular chaperons present in
all kingdoms and their role in the endoplasmic reticulum is to transiently bind to
unfolded proteins to prevent intramolecular and intermolecular interactions that can
result in permanent misfolding or aggregation, with the subsequent loss of their function
(Gething and Sambrook 1992; Hendershot et al. 1996). A gene encoding for a BiP
protein has been described in G. intraradices but no information is available on its
possible biological function, although it was postulated that it could be similar to that of
animals or plants (Porcel et al. 2007).
In this study, a strain of G. intraradices isolated from Cabo de Gata Natural Park
(Almería, Spain), a region with serious problems of salinity and affected by
desertification, has been compared with a collection strain, used as a model fungus.
Bago et al. (1998b) highlighted monoxenic cultures as an appropriate tool for studying
the extraradical phase of AM symbiosis. In the present work we took advantage of the
in vitro monoxenic culture of AM fungi in order to study under increasing salinity
levels the differences in development and in expression of putative stress-responsive
genes of two strains of the AM fungus G. intraradices. In a parallel study, we
conducted an in vivo experiment with the same AM fungi and a host plant of agronomic
importance such as maize, in order to test their symbiotic efficiencies under increasing
salinity levels.
Identification of the mycorrhizal strain isolated from Cabo de Gata Natural Park
AM fungal spores were separated from the soil samples by a wet sieving process
(Sieverding 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-
glycerine (PVLG) (Koske and Tessier 1983); a mixture of PVLG and Melzer’s reagent
(Brundrett et al. 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent; and
water (Spain 1990). For identification of the AMF species, spores were then examined
using a compound microscope at up to 400-fold magnification as described by the
glomeromycotean classification of Oehl et al. (2011) recently published in the new
journal IMA Fungus. The species was clearly identified based on its spore morphology
as Glomus intraradices (Schenk and Smith 1982), which has been recently reassigned to
Rhizophagus intraradices (N.C. Schenck and G.S. Sm.) C. Walker & A. Schuessler
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Chapter 3
2010. Spores presented a globose form, from 65 to 145 μm diameter, and light colour
between white and yellow (some old spores had a brownish colour). Up to four layers
were observed in some of the samples and some of them presented a hyphal constriction
at the base of the spore.
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures were surface-sterilized
with chloramine T (2%) and streptomycin (0.02%) and crushed with a sterile disposable
micropestle in 40 μL milli-Q water (Ferrol et al. 2004). A two-step PCR was conducted
to amplify the AM fungal DNA from the spores. The first PCR step was performed with
the universal eukaryote primers NS1 and NS4 region of the small subunit ribosomal
gene and the second with the specific AM fungal primers AML1 and AML2 (Lee et al.
2008). The amplified DNA was purified using the Ilustra™ GFX™ PCR DNA and Gel
Band Purification Kit (GE Helthcare, UK). DNA fragments were sequenced on an
automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence data were
compared to gene libraries (EMBL and GenBank) using BLAST program (Altschul et
al. 1990).
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our G. intraradices CdG strain, with sequence accession number
FR750209 (Kruger et al. 2012) having a 99% identity.
The AM fungal strain has been incorporated to the collection of Zaidin
Experimental Station, Granada, Spain, under accession EEZ 195.
The two Glomus intraradices strains used in this study were established in
monoxenic culture as described by St-Arnaud et al. (1996). For that, the clone DC2 of
carrot (Daucus carota L.) Ri-T DNA transformed roots were cultured in two-
compartment Petri dishes with the AM fungal strain DAOM 197198 of G. intraradices
Smith and Schenck [recently reassigned to G. irregulare by Stockinger et al. (2009) and
then as Rhizophagus irregularis (Błaszk., Wubet, Renker and Buscot) C. Walker and A.
Schüßler comb. nov.] or with the strain G. intraradices isolated from Cabo de Gata
(CdG) Natural Park in Almería (Southeast Spain; located at 36°44´41´´N,
02°07´26´´W).
The root compartment of each plate was filled with sterile minimal medium, as
described by Chabot et al. (1992) referred to as “M” throughout this report, to the top of
the division wall. The medium had been autoclaved for 20 minutes at 120ºC. The hyfal
compartment of each plate was filled in the same manner except that M medium used
did not contain any sucrose (referred to as “M-C” in this work). In addition, prior to the
sterilization of the M-C medium NaCl had been added to the medium to obtain 0, 75 or
150 mM concentrations of salt. The media were allowed to solidify at room
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Chapter 3
temperature. There were six different treatments, with 50 replicate plates per treatment,
totalling 300 plates.
A piece of M agar (approximately 0.5 cm2) was cut from the root compartment
of each Petri dish and replaced with fungal inoculum, which consisted of a piece of agar
of the same size obtained from stock monoxenic cultures containing spores and hyphae
of the collection G. intraradices strain or the strain G. intraradices CdG. The
specimens, kept in continuous monoxenic cultures, were provided by the culture
collections of Zaidin Experimental Station, Granada, Spain. Three to four pieces of
transformed carrot roots (2.5 cm length each), grown in M medium, were placed on top
of the fungal inoculum in the root compartments. Two weeks after inoculation plates
were checked and if roots were crossing onto the distal compartments, they were
aseptically moved back to their proximal compartments. This check was subsequently
repeated once a week along the experiment. The plates were incubated in the dark at
24ºC for 2 months.
Parameters measured
Four, six and eight weeks after inoculation plates were examined under
dissecting microscope (Nikon, SMZ 1000, Japan) and AM fungal development was
assessed using the method described by Marsh (1971) and modified by Bago and Cano
(2005). A transparent 2 mm grid was used to determine the hyphal length, the number
of branched absorbing structures (BAS) (Bago et al. 1998a) and the number of spores in
three areas of 1 cm2 per distal compartment of each plate.
After eight weeks, the mycelium from the distal compartment was isolated.
Citric acid monohydrate 10 mM at pH 6 was used to extract the mycelium from the M-
C medium. Then it was immersed in liquid nitrogen and stored at -80ºC until RNA was
extracted using the RNeasy plant mini kit (Qiagen, Valencia, CA, U.S.A.).
First single-strand cDNA was primed by random hexamers using 100–1,000 ng
of DNase-treated RNA. RNA samples were denatured at 65ºC for 5 min and then
reverse transcribed at 25ºC for 10 min and 42ºC for 50 min in a final volume of 20 μl
containing 10 μl of total RNA, 10 μM random primers (Invitrogen, Carlsbad, CA,
USA), 0.5 mM dNTPs, 10 U RNase inhibitor, 4 μl of 5x buffer, 2 μl 0.1 M DTT, and 1
μl of Superscript II Reverse Transcriptase (Invitrogen). The samples were precipitated
with 1 (v/v) isopropanol and suspended in 20 μl of water.
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Chapter 3
Statistical Analysis
Statistical analysis was performed using SPSS 19.0 statistical program (SPSS
Inc., Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey
test with P< 0.05 as the significance cut-off.
In vivo experiment
94
Chapter 3
AM fungal strain G. intraradices isolated from Cabo de Gata (CdG) and (3) plant
inoculated with the model AM fungus G. intraradices reproduced at collection of the
Zaidin Experimental Station. There were 30 replicates of each inoculation treatment,
totalling 90 pots (one plant per pot), so that ten pots of each microbial treatment were
grown under nonsaline conditions throughout the entire experiment, while ten pots per
treatment were subjected to 66 mM of NaCl and the remaining ten pots per treatment
were subjected to 100 mM of NaCl.
Loamy soil was collected from Granada province (Spain, 36º59’34’’N;
3º34’47’’W), sieved (5 mm), diluted with quartz-sand (<2 mm) (1:1, soil:sand, v/v) and
sterilized by steaming (100ºC for 1 h on 3 consecutive days). The original soil had a pH
of 8.2 [measured in water 1:5 (w/v)]; 1.5 % organic matter, nutrient concentrations (g
kg-1): N, 1.9; P, 1 (NaHCO3-extractable P); K, 6.9. The electrical conductivity of the
original soil was 0.5 dS m-1.
Three seeds of maize (Zea mays. L) were sown in pots containing 900 g of the
same soil/sand mixture as described above and thinned to one seedling per pot after
emergence.
Mycorrhizal inoculum was bulked in an open-pot culture of Zea mays L. and
consisted of soil, spores, mycelia and infected root fragments. The AM species used
were two strains of Glomus intraradices, the first one from our culture collection and
the second one isolated from Cabo de Gata (Almería, Spain). Appropriate amounts of
each inoculum containing about 700 infective propagules (according to the most
probable number test), were added to the corresponding pots at sowing time just below
maize seeds.
Uninoculated control plants received the same amount of autoclaved
mycorrhizal inocula together with a 10 ml aliquot of a filtrate (< 20 μm) of the AM
inocula in order to provide a general microbial population free of AM propagules.
Growth conditions
The experiment was carried out under glasshouse conditions with temperatures
ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 70-80%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 45 days
prior to salinization to allow adequate plant growth and symbiotic establishment prior to
application of the salt stress. Three concentrations (0, 66, and 100 mM NaCl) of saline
solution were reached in the soil substrate by adding appropriate dilutions of a stock 2
M saline solution. The concentration of NaCl in the soil was increased gradually on
alternative days to avoid an osmotic shock. It took 8 days, to reach the desired 66 and
100 mM NaCl levels. The electrical conductivities in the soil were 0.25, 6.9 and 9.3 dS
95
Chapter 3
m-1 for the salt levels of 0, 66, and 100 mM NaCl, respectively. Plants were maintained
under these conditions for additional 30 days.
Symbiotic development
Biomass production
At harvest (75 days after planting), the shoot and root system were separated and
the shoot dry weight (SDW) and root dry weight (RDW) were measured after drying in
a forced hot-air oven at 70ºC for two days.
Photosynthetic efficiency
Stomatal conductance
Stomatal conductance was measured two hours after light turned on by using a
porometer system (Porometer AP4, Delta-T Devices Ltd, Cambridge, UK) following
the user manual instructions. Stomatal conductance measurements were taken in the
third youngest leaf from five different plants from each treatment.
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Chapter 3
Results
Experiment in vitro
After four week of culture, the hyphal length produced by both AM fungal
strains was negatively affected by the presence of 75 and 150 mM NaCl in the medium
(Figure 1A). The effect of salinity decreasing hyphal length was similar for the two
fungal strains. At six weeks, only 150 mM NaCl decreased significantly the hyphal
length of both fungal strains as compared to the control treatment without salt (Figure
1B). Again, both AM fungal strains showed a similar hyphal length decrease. Finally,
after eight weeks of culture, Gi CdG did not show hyphal length reduction in response
to any of the salt levels applied to the medium (Figure 1C). In contrast, the collection G.
intraradices strain decreased the hyphal length after addition of 75 mM NaCl (26% of
decrease) and 150 mM NaCl (37% of decrease).
The number of spores produced by both fungal strains increased transiently at
four weeks after the application of 75 mM NaCl, while under 150 mM NaCl, it showed
similar levels than those of the no stress treatment (Figure 2A). At six weeks Gi CdG
showed no effect of salt on the number of spores produced, while the collection G.
intraradices strain increased significantly the number of spores produced after the
addition of 150 mM NaCl (Figure 2B). Finally, at eight weeks, both fungal strains
enhanced the number of spores produced after the addition of either 75 or 150 mM
NaCl, with no significant differences between both strains (Figure 2C).
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Chapter 3
1500 Gi CdG A
1200
Gi collect
900
a a
600
300 b b b b
0
0 mM 75 mM 150 mM
1500 B
Hyphal lenght (cm)
1200 a ab
abc bc bc
900 c
600
300
0
0 mM 75 mM 150 mM
1500 a a a C
ab
1200
bc
900 c
600
300
0
0 mM 75 mM 150 mM
Figure 1. Total hyphal length (cm) formed in the hyphal compartment by two G. intraradices strains
grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain Gi CdG
and grey bars represent the collection G. intraradices strain. Measurements were done after 4 weeks (A),
6 weeks (B) or 8 weeks (C) of fungal growth in the medium. Means followed by different letters are
significantly different (P<0.05).
100 Gi CdG
A
80
Gi collect
60
40
20 ab a
b b b b
0
00mM
mM 75
75mM
mM 150
150 mM
mM
100
B
Number of spores
80
60 a
40 b
b b b
20
b
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM
100 a C
a a a
80
60 b
40 b
20
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM
Figure 2. Total number of spores formed in the hyphal compartment by two G. intraradices strains
grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain Gi
CdG and grey bars represent the collection G. intraradices strain. Measurements were done after 4 weeks
(A), 6 weeks (B) or 8 weeks (C) of fungal growth in the medium. Means followed by different letters are
significantly different (P<0.05).
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Chapter 3
After four weeks of culture, the number of BAS produced was negatively
affected by the highest salt level (150 mM) applied, with a similar decrease of this
parameter in both fungal strains (Figure 3A). At week 6, salinity influenced differently
the number of BAS produced by each fungal strain (Figure 3B). Thus, in Gi CdG, this
parameter was decreased by 150 mM NaCl, while the collection G. intraradices strain
did not show significant changes as a consequence of salt application. However, after
eight weeks of fungal culture no significant differences in BAS production were
observed among treatments (Figure 3C).
Gi CdG
90 Gi collect A
60 a a
ab
30 ab
b b
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM
90
a ab B
ab a
Number of BAS
60
ab b
30
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM
90 a C
a a
60 a a
a
30
0
00 mM
mM 75
75 mM
mM 150
150 mM
mM
Figure 3. Total number of BAS formed in the hyphal compartment by two G. intraradices strains grown
in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain Gi CdG and
grey bars represent the collection G. intraradices strain. Measurements were done after 4 weeks (A), 6
weeks (B) or 8 weeks (C) of fungal growth in the medium. Means followed by different letters are
significantly different (P<0.05).
The most remarkable results were those related to the expression of the two
chaperone-encoding genes Gint14-3-3 and GintBIP (Figures 4 and 5). The expression of
both genes was considerably higher in Gi CdG than in the collection G. intraradices
strain, even in absence of NaCl in the growing medium (up regulation by 85 fold
Gint14-3-3 and by 96 fold GintBIP). The presence of salt in the medium further up
regulated the expression of these two genes in Gi CdG, mainly at 150 mM NaCl (up
regulation of Gint14-3-3 by 9 fold as compared to 0 mM NaCl or up regulation of
GintBIP by 13 fold as compared to 0 mM NaCl). In contrast, in the case of the
collection G. intraradices strain, only the application of 75 mM NaCl up regulated the
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Chapter 3
expression of this gene, while the application of 150 mM NaCl down regulated or kept
unchanged the expression of Gint14-3-3 and GintBIP genes, respectively.
260 Gi CdG
a
Gi collect
210
160
110
Gint14-3-3 Relative expression
b
60
c
10
1,0
0,9
0,8
0,7
0,6
0,5 d
0,4
e
0,3
f
0,2
0,1
0,0
0 mM 75 mM 150 mM
Figure 4. Analysis of Gint14-3-3 gene expression by real time quantitative RT-PCR in two G.
intraradices strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars
represents strain Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by
different letters are significantly different (P<0.05).
Gi CdG a
30,20 Gi collect
25,20
20,20
15,20 b
GintBiP Relative expression
10,20
5,20 c
0,20
0,10
0,09
0,08
0,07 d
0,06
0,05
0,04 e
0,03 e
0,02
0,01
0,00
0 mM 75 mM 150 mM
Figure 5. Analysis of GintBiP gene expression by real time quantitative RT-PCR in two G. intraradices
strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars represents strain
Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by different letters
are significantly different (P<0.05).
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Chapter 3
0,93 Gi CdG
a
0,83 Gi collect
0,73
GintSOD1 Relative expression
0,63
0,53
0,43
0,33
b
0,23 b b
0,13
0,03
0,030
0,025
0,020
0,015
0,010
0,005 c c
0,000
0 mM 75 mM 150 mM
Figure 6. Analysis of GintSOD1 gene expression by real time quantitative RT-PCR in two G.
intraradices strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars
represents strain Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by
different letters are significantly different (P<0.05).
The expression of the GintAQP1 gene in Gi CdG and collection strains increased
alter the application of 150 and 75 mM NaCl, respectively. In the collection strain
however, addition of 150 mM NaCl to the culture medium caused no significant effect
on the expresión of this gene (Figure 7). At 75 mM Na Cl the expression of GintAQP1
was higher in the collection G. intraradices strain than in Gi CdG, while at 150 mM
NaCl it was just the opposite.
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Chapter 3
Figure 7. Analysis of GintAQP1 gene expression by real time quantitative RT-PCR in two G.
intraradices strains grown in monoxenic culture and subjected to 0, 75 or 150 mM NaCl. White bars
represents strain Gi CdG and grey bars represent the collection G. intraradices strain. Means followed by
different letters are significantly different (P<0.05).
Experiment in vivo
Salt stress did not affect significantly SDW in non-AM plants, while both AM
treatments reduced SDW at 100 mM NaCl as compared to the non-salt stressed
treatment (Figure 8A). In any case, plants inoculated with Gi CdG exhibited the highest
SDW production at all salt levels studied. Indeed, the increases in SDW at 0 and 100
mM NaCl were, respectively, 26 and 17%, as compared to non-AM plants. Plants
inoculated with the collection G. intraradices strain exhibited similar SDW values than
non-AM plants at all salt levels.
The RDW showed no significant changes as a consequence of either salt levels
applied or the AM fungal strain inoculated (Figure 8B).
Symbiotic development
102
Chapter 3
1
0
0 mM 66 mM 100 mM
Root dry weight (g.plant-1)
2,5 a B
a ab
2 ab ab ab ab
ab b
1,5
0,5
0
0 mM 66 mM 100 mM
-1
Figure 8. Shoot (A) and root (B) dry weights (g plant ) in maize plants. Black bars represent
noninoculated control plants (NM), grey bars represent plants inoculated with the collection G.
intraradices strain and white bars represent plants inoculated with the native strain Gi CdG. Plants were
subjected to 0, 66 or 100 mM NaCl. Columns with different letters are significantly different (P<0.05).
25
Electrolyte Leakage (%)
a
B
A
20 ab ab ab
ab ab
15 c c c
10
5
0
0 mM 66 mM 100 mM
(%)
100 a B
a
AM root colonization
80 b
60
%
40 c
c c
20
0
0 mM 66 mM 100 mM
Figure 9. Electrolyte leakage (A) and percentage of mycorrhizal root length (B) in maize plants. Black
bars represent noninoculated control plants (NM), grey bars represent plants inoculated with the
collection G. intraradices strain and white bars represent plants inoculated with the native strain Gi CdG.
Plants were subjected to 0, 66 or 100 mM NaCl. Columns with different letters are significantly different
(P<0.05).
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Chapter 3
The applied salt stress did not significantly increase the relative electrolyte
leakage in maize plants from any treatment (Figure 9A). Under non saline conditions,
plants colonized by Gi CdG exhibited 25% less electrolyte leakage than non-AM plants,
while no significant effect was observed in plants inoculated with the collection G.
intraradices strain. At 66 mM NaCl, results were similar than under non saline
conditions. Finally, at 100 mM NaCl both AM fungi decreased electrolyte leakage as
compared to non-AM plants. In fact, plants inoculated with the collection G.
intraradices strain decreased this parameter by 22% and those inoculated with Gi CdG
did it by 36%.
Stomatal conductance
Photosystem II efficiency
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Chapter 3
30,00
30,00 ab a
Stomatal conductance 25,00
25,00 a A
(mmolH2Om-2s-1)
20,00
20,00 b
bc
bc bc b
15,00
15,00 cd
cd
d
10,00
10,00 NM
Gi collect
5,00
5,00 Gi CdG
0,00
0,00
00 mM
mM 66
66 mM
mM 100 mM
100 mM
Photosynthetic efficiency
0,7 a
ab ab
ab B
0,6 b ab b
0,5 c
0,4 d
0,3
0,2
0,1
0
0 mM 66 mM 100 mM
-2 -1
Figure 10. Stomatal conductance (mmol H2O m s ) (A) and efficiency of photosystem II (B) in maize
plants. Black bars represent noninoculated control plants (NM), grey bars represent plants inoculated
with the collection G. intraradices strain and white bars represent plants inoculated with the native strain
Gi CdG. Plants were subjected to 0, 66 or 100 mM NaCl. Columns with different letters are significantly
different (P<0.05).
Discussion
105
Chapter 3
Salt stress inhibits plant photosynthetic ability, which leads to a decrease in crop
production (Pitman and Läuchli 2002), but several publications report that AM fungi in
saline soils can decrease plant yield losses by increasing their photosynthetic capacity
(Reviewed by Evelin et al. 2009; Ruiz-Lozano et al. 2012). This agrees with results
obtained in this work with maize plants inoculated with Gi CdG. Moreover, plants
inoculated with the collection G. intraradices strain had higher percentage of root
colonization than those inoculated with Gi CdG, stressing that the symbiotic efficiency
of Gi CdG in terms of plant growth is higher than that of the collection G. intraradices.
The higher symbiotic efficiency of Gi CdG was also corroborated by the enhanced
efficiency of photosystem II and stomatal conductance and the lower electrolyte leakage
exhibited by maize plants under the different conditions assayed. This agrees with
previous reports that found higher photosynthetic efficiency in leaves of mycorrhizal
plants under saline conditions (Sheng et al. 2008; Zuccarini and Okurowska 2008),
higher stomatal conductance (Ruíz-Lozano et al. 1996; Jahromi et al. 2008; Sheng et al.
2008) and improved integrity and stability of the cellular membranes (Feng et al. 2002;
Garg and Manchanda 2008; Kaya et al. 2009). Stahl and Smith (1984) reported that
Agropyron smithii colonized with G. microcarpum collected from a desert had
increased stomatal opening under arid condition than that colonized with G.
microcarpum collected from a more mesic site. This is an example to show a specific
AM fungal strain being more adapted to specific environmental condition.
The presence of salts in the growth medium may induce changes in the length
and other morphological properties of the hyphae, thus affecting their symbiotic
efficiency and also their infective capacity. Indeed, some studies state that salt inhibits
spore germination or other fungal propagules, colonization of the plant roots and
sporulation of AM fungi (Juniper and Abbott 2006; Giri et al. 2007; Sheng et al. 2008;
Jahromi et al. 2008). In this study, the collection G. intraradices strain showed a
transient enhancement in the number of spores and BAS structures at 6 weeks after
growing, with no significant differences at 8 weeks. This could be regarded as a
symptom of stress perception in this fungal strain, because spores are a form of
resistance propagules that can survive under adverse conditions and BAS are thought to
be associated with the formation of spores (Bago et al. 1998a). In contrast, the hyphal
length was reduced in both fungal strains by salt application, but at 8 weeks after
growing this decrease was significantly higher in the collection G. intraradices than in
Gi CdG. As the mycelium is not a form of resistance propagule in AM fungi, a higher
hyphal development can be considered in terms of tolerance, being the AM fungus from
Cabo de Gata a more tolerant strain than the collection G. intraradices. Previous reports
have indicated that hyphal networks are a very important source for the rapid initiation
of root colonization (McGee et al. 1997; Smith and Read 2008). The maintenance of
AM fungi in ecosystems is dependent on the persistence of a potential inoculum in soils
(Brundrett 1991). Carvalho et al. (2004) found evidence for potential adaptation of
indigenous AM fungi to salt marsh conditions and for the ability of different propagules
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Chapter 3
of these fungi to colonize new plants and spread the infection through the roots. Brito et
al. (2011) also showed that extraradical mycelium of native AM fungi can survive the
dry and hot summer in a typical Mediterranean region and initiate colonization of wheat
plants at the onset of the growing season; the same may occur with Gi CdG.
To determine the possible causes of the different behaviour and tolerance of both
AM fungal strains under saline conditions, we evaluated the effects of salinity on
several fungal genes potentially involved in the response to salinity. The induction of
genes encoding for chaperones, ROS scavengers, as well as, water channels is important
to re-establish cellular homeostasis and membranes stability during stresses (Xiong and
Zhu 2002; Bhatnagar-Mathur et al. 2008).
All the genes studied were up-regulated by increasing salinity in the fungus
isolated from Cabo de Gata, while the collection G. intraradices strain showed only an
up-regulation of the GintSOD1 gene. The overexpression of these genes under saline
conditions indicates that they have a role in the response of the fungus against osmotic
stress. Indeed, chaperone-like proteins, such as 14-3-3 and BiPs, have been
demonstrated to confer tolerance to a variety of stresses in plants, while in fungi the
literature is scarce. It has been proposed that BiP overexpression may prevent the cell
from sensing osmotic stress-induced variations in ER function by keeping ER basic
activities to a normal level under saline conditions (Valente et al. 2009). This is because
protein folding in the ER is facilitated by molecular chaperones, which prevent
nonproductive intermolecular interactions of folding intermediates and subsequent
misaggregation of proteins within the lumen of the ER (Hammond and Helenius 1995).
The AM fungal gene GintBIP, was studied in vitro by Porcel et al. (2007): they added
25% of PEG to the medium and the GintBIP gene expression increased by 41%. When
the gene was analyzed in vivo using maize, soybean and tobacco plants inoculated with
G. intraradices, the gene showed even higher expression. This was concomitant with
improved tolerance to drought (Porcel et al. 2007).
In a previous study, Porcel et al. (2006) found that the addition of PEG to the
medium increased Gint14-3-3 gene expression by 1200%. Expression of the gene was
also up-regulated in roots of mycorrhizal maize, lettuce and tobacco but not in soybean
where it did not show any change. It was proposed that Gint14-3-3 protein could
regulate the activity of plasma membrane H+-ATPases of either the fungus or the host
plant, to activate its pumping activity, which is essential to cope with osmotic stress
(Palmgren 1998). Indeed, the activity of plasma membrane H+-ATPase is highly
regulated by factors that affect the cell physiology, including stress conditions and
enhanced ATPase activity is crucial for the protective system that different organisms
have developed against external adverse influence (Palmgren 1998). Moreover, as 14-3-
3 proteins are found in association with key control enzymes of primary metabolism, its
overexpression could rapidly alter metabolic flux in response to signals such as salt
stress (Finnie et al. 1999) and they could also regulate the expression of stress-inducible
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Chapter 3
genes by regulating the activity and or localization of transcription factors (Muslin and
Xing 2000).
Like other abiotic stresses, salinity also induces oxidative stress in plants
(Hajiboland and Joudmand 2009). In the field of the AM symbiosis, several studies
suggested that AM symbiosis helps plants to alleviate salt stress by enhancing the
activities of antioxidant enzymes (Alguacil et al. 2003; Zhong Qun et al. 2007; Garg
and Manchanda 2009; Talaat and Shawky 2011), but the response of the individual
enzymes varies with respect to the host plant and the fungal species involved in the
association. From the fungal side, González-Guerrero et al. (2010) described a
GintSOD1 gene encoding a functional protein that scavenges ROS. The up-regulation of
GintSOD1 transcripts in the fungal mycelia treated with paraquat and Cu indicated that
the gene product might be involved in the detoxification of the ROS induced by these
two external agents. Lanfranco et al. (2005) described and orthologous gene of
Gigaspora margarita, which may play a pivotal role in the relationship of the fungus
with its host plant, as it has been described in the ericoid mycorrhizal fungus
Oidiodendron maius (Abbà et al. 2009). As far as we know, this is the first study on the
effect of salinity on the expression of GintSOD1, showing that the gene is up-regulated
under saline conditions and providing evidence for a role of GintSOD1 in the fungal
response to the oxidative stress induced by salinity. In any case, the up-regulation of
this gene was lower in Gi CdG, suggesting that under salinity the accumulation of ROS
by this fungal strain could be lower than by the collection G. intraradices strain.
Salinity decreases the water potential of the medium, hampering the uptake of
water from the growing medium. Thus, the activity of aquaporins should be important
to living organisms in order to cope with the water deficit induced by salt stress.
Although it is well known that mycorrhizal mycelium transports water from the soil to
the roots, only three reports have studied mycorrhizal fungal aquaporins (Aroca et al.
2009; Dietz et al. 2011; Navarro-Ródenas et al. 2012). Dietz et al. (2011) reported that,
in the aquaporin gene family of Laccaria bicolor, three out of seven L. bicolor
membrane intrinsic proteins showed high water permeability and two of them were also
found to increase ammonia transport. Navarro-Ródenas et al. (2012) found high levels
of water conductivity of TcAQP1 that could be related to the adaptation of Terfezia
claveryi to semiarid areas because, as it was shown in a previous study, the mycelium of
this mycorrhizal fungus exhibited drought tolerance under in vitro conditions (Navarro-
Ródenas et al. 2012). However, only one study has been done so far on aquaporins from
an AM fungus (Aroca et al. 2009). Authors found some evidences supporting the idea
that fungal aquaporins could compensate the down regulation of host plant aquaporins
caused by osmotic stress. They also found that GintAQP1 expression was up regulated
in the osmotically non-stressed part of the mycelium when the other mycelium part was
stressed by NaCl. In the present study we found an up-regulation of GintAQP1 gene at
75 mM NaCl in the isolate from collection, but not in Gi CdG. In contrast, at the highest
salinity level (150 mM NaCl) the up regulation was found only in Gi CdG. Thus, Gi
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CdG has the ability to induce the expression of this aquaporin gene when the salt in the
medium reaches high levels. The biological significance of the up-regulation of
GintAQP1 gene remains to be elucidated since it was not possible to demonstrate
whether the respective aquaporin protein indeed transport water or other substrates
(Aroca et al. 2009).
In conclusion, results from this study show that the strain Gi CdG exhibited a
higher tolerance to salinity than the collection G. intraradices strain and grew and
developed better under saline conditions. The present study demonstrates that the Gi
CdG strain exhibits a better symbiotic efficiency in an already established symbiosis
under conditions of salt stress. These effects could be due to a fungal adaptation to the
saline environment where the fungus was isolated. The adaptation to salinity may be
related to the significant up-regulation of genes with chaperone activity or genes
encoding for aquaporins. The fungus from Cabo de Gata may reduce the production of
ROS, which in turns was evidenced by a lower induction of GintSOD1 gene.
The present study underlines the importance of salt adaptation in AM fungi.
Stress tolerance can only be gained through long-term exposure to chronic stress. Thus
AM fungi isolated from areas affected by salinity will be a powerful strategy to enhance
the tolerance of crops to saline stress conditions or in revegetation programs of
degraded areas affected by osmotic environmental constrains.
Acknowledgements
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CAPÍTULO 4
CHAPTER 4
Resumen
121
122
Chapter 4
Beatriz Estrada1, Ricardo Aroca1, Frans J.M. Maathuis2, José Miguel Barea1 and
Juan Manuel Ruiz-Lozano1*
1
Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación
Experimental del Zaidín (CSIC). Profesor Albareda nº 1, 18008 Granada, Spain.
2
Department of Biology Area 9, University of York, York YO10 5DD, UK
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Abstract
Soil salinity seriously restricts plant growth and productivity. Maize is a salt-sensitive
plant, where Na+ represents the major ion causing toxicity because it can compete with
K+ for binding sites at the plasma membrane. Inoculation with arbuscular mycorrhizal
fungi (AMF) can alleviate salt stress in several host plants through several mechanisms.
These may include ion selection during the fungal uptake of nutrients from the soil or
during transfer to the host plant. It has been proposed that AM benefits could be
enhanced when native AMF isolates, physiologically and genetically adapted to the
stress conditions of their environment, are used. In this study we investigated whether
native AMF isolated from Cabo de Gata Natural Park (CdG) (an area with serious
problems of salinity and affected by desertification) can help maize plants to overcome
the negative effects of salinity stress better than non-AM plants or plants inoculated
with non-native AMF. Indeed, results showed that maize plants inoculated with two out
the three native AMF had the highest shoot dry biomass at all salinity levels, while
biomass in plants inoculated with the collection AM strain was similar to the non-
mycorrhizal plants. Plants inoculated with the three native AMF showed significant
increase of K+ and reduced Na+ accumulation as compared to non-mycorrhizal plants,
concomitantly with higher K+/Na+ ratios in their tissues. These effects correlated with
regulation of ZmAKT2, ZmSOS1 and ZmSKOR genes expression in the roots of maize
plants colonized by native AMF, contributing to K+ and Na+ homeostasis.
Key words: adaptation, ion homeostasis, native arbuscular mycorrhizal fungi, salinity,
tolerance
Introduction
Salinity is a major and increasing problem which restricts plant growth and
productivity. More than 800 million hectares of land throughout the world are salt
affected (including both saline and sodic soils) (FAO, 2005). This is over 6% of the
total land area of the world. High amounts of salts in soils are responsible for yield
reduction in one third of the global arable land (Lambers, 2003). This is particularly the
case in regions with high rates of evaporation, like arid and semiarid areas (Hammer et
al., 2011). Most crops are glycophytic and tolerate salinity to a threshold level. Above
this level, yield decreases (Khan et al., 2006), since excess of salt inhibits
photosynthetic ability and induces physiological drought in plants (Pitman and Läuchli,
2002). Maize (Zea mays L.) is classified as a salt-sensitive plant (Maas and Hoffman,
1977). Although maize is originally from Mesoamerica, nowadays it is the third most
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important cereal crop and ranks first in countries with developing economies (Mejía,
2003).
Most glycophytes tolerate salinity by restricting the uptake of Na+ and Cl- while
maintaining uptake of macronutrients such as K+ or N (Teakle and Tyerman, 2010).
Although Cl- is considered an essential micronutrient for higher plants involved in the
regulation of important cellular functions such as enzyme activity, maintenance of
membrane potentials, and as a co-factor in photosynthesis and pH gradients (White and
Broadley, 2001), it can be toxic to plants at high concentrations (Xu et al., 2000).
However for maize, it has been shown that Na+ (and not Cl-) represents the major ion
causing toxicity related to salinity (Fortmeier and Schubert, 1995) because it can
compete with K+ for binding sites at the plasma membrane. The K+ ion is essential for
protein synthesis, activation of many enzymes and photosynthesis and it plays a central
role in osmotic adjustment, turgor maintenance, and in the control of stomata opening
(Maathuis and Amtmann, 1999). It has been shown that chloroplast function is impaired
when K+ is displaced by Na+, leading to uncontrolled water losses (Slabu et al., 2009).
Furthermore, adequate K+ is very important to maintain cytosolic ion homeostasis in
Na+-stressed plants (Zhu, 2003), a function which is disrupted by excessive Na+ entry
(Demidchik and Maathuis, 2007). Accumulation of Na+ and impairment of K+ nutrition
is a major characteristic of salt stressed plants, the mechanisms of which are only
partially understood. However, salt stress often causes reduction in plant tissue K+
content, and the K+/Na+ ratio is considered a useful parameter to assess salt tolerance
(Maathuis and Amtmann, 1999; Chen et al., 2007). Another important response of
glycophytes to salinity stress, associated with osmoregulation adjustment, is the
accumulation of osmotically active organic solutes such as proline and glycine-betaine
(Munns, 2005). Proline maintains the osmotic balance and protects enzymes in presence
of high cytoplasmic electrolyte concentrations (Greenway and Munns, 1980; Hajlaoui et
al., 2010). However, the significance of proline accumulation in osmotic adjustment is
still debated and varies according to the species (Lutts et al., 1996; Rodriguez et al.,
1997).
Plants can overcome salinity effects by interacting with several beneficial soil
microorganisms. Soil microbiota, such as arbuscular mycorrhiza fungi (AMF) live
symbiotically associated with the roots of 80% of terrestrial plants (Smith and Read,
2008) and are able to increase plant growth and crop productivity under different
environmental stresses (Barea et al., 2012). Several studies have shown that inoculation
with AMF can alleviate salt stress (Sannazzaro et al., 2006; Jahromi et al., 2008;
Estrada et al., 2012). Improved salt tolerance following mycorrhizal colonization may
be the result of a more efficient nutrient uptake (Cantrell and Linderman, 2001), ion
balance (Giri et al., 2007), protection of enzyme activities (Rabie and Almadini, 2005),
increase in photosynthesis ability (Sheng et al., 2008) and facilitation of water uptake in
plants (Aroca et al., 2007).
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transporters, we also analyzed the regulation by these AMF of key plant ion transporters
expected to be affected by salinity.
Identification of the mycorrhizal strains isolated from Cabo de Gata Natural Park
AM fungal spores were separated from the soil samples by a wet sieving process
(Sieverding, 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-
glycerine (PVLG) (Koske and Tessier, 1983); a mixture of PVLG and Melzer’s reagent
(Brundrett et al., 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent; and
water (Spain, 1990). For identification of the AMF species, spores were then examined
using a compound microscope at up to 400-fold magnification as described for
glomeromycotean classification by Oehl et al. (2011). The species were identified based
on its spore morphology as a Rhizophagus intraradices (Schenk and Smith, 1982),
Claroideoglomus etunicatum (Becker and Gerdemann, 1977) and Septoglomus
constrictum (Trappe, 1977).
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures of each fungal strain
were surface-sterilized with chloramine T (2%) and streptomycin (0.02%) and crushed
with a sterile disposable micropestle in 40 μL milli-Q water (Ferrol et al., 2004). A two-
step PCR was conducted to amplify the AM fungal DNA from the spores. The first PCR
step was performed with the universal eukaryote primers NS1 and NS4 region of the
small subunit ribosomal gene and the second with the specific AM fungal primers
AML1 and AML2 (Lee et al., 2008). The amplified DNA was purified using the
Ilustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Helthcare, UK). DNA
fragments were sequenced on an automated DNA sequencer (Perkin-Elmer ABI Prism
373). Sequence data were compared to gene libraries (EMBL and GenBank) using
BLAST program (Altschul et al., 1990).
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our Rhizophagus intraradices CdG strain, with sequence accession
number FR750209 (Krüger et al., 2012) having a 99% identity. Septoglomus
constrictum was the closest relative to our Se. constrictum CdG strain, with sequence
accession number FR750212 (Krüger et al., 2012) having a 99% identity. Finally,
Claroideoglomus etunicatum was the closest relative of our Cl. etunicatum CdG strain,
with sequence accession number FR750216.1 (Krüger et al., 2012) having also a 99%
identity. The AM fungal strains have been incorporated to the collection of Zaidin
Experimental Station, Granada, Spain, under accession numbers EEZ 195, EEZ 196 and
EEZ 163, respectively.
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Experimental design
The experiment consisted of a randomized complete block design with five inoculation
treatments: (1) non-mycorrhizal control plants, (2) plants inoculated with the model AM
fungus Rh. intraradices (Ri collect), reproduced at collection of the Zaidin
Experimental Station, (3) plants inoculated with the AM fungal strain Rh. intraradices
isolated from Cabo de Gata Natural Park (Ri CdG), (4) plants inoculated with the AM
fungal strain Se. constrictum isolated from CdG (Sc CdG) and (5) plants inoculated with
the AM fungal strain Cl. etunicatum isolated from CdG (Ce CdG). There were 30
replicates of each inoculation treatment, totalling 150 pots (one plant per pot), so that
ten of each microbial treatment were grown under nonsaline conditions throughout the
entire experiment, while ten pots per treatment were subjected to 66 mM of NaCl and
the remaining ten pots per treatment were subjected to 100 mM of NaCl.
Loamy soil was collected from Granada province (Spain, 36º59’34’’N; 3º34’47’’W),
sieved (5 mm), diluted with quartz-sand (<2 mm) (1:1, soil:sand, v/v) and sterilized by
steaming (100ºC for 1 h on 3 consecutive days). The original soil had a pH of 8.2
[measured in water 1:5 (w/v)]; 1.5 % organic matter, nutrient concentrations (g kg-1): N,
1.9; P, 1 (NaHCO3-extractable P); K, 6.9. The electrical conductivity of the original soil
was 0.5 dS m-1.
Three seeds of maize (Zea mays. L) were sown in pots containing 900 g of the
same soil/sand mixture as described above and thinned to one seedling per pot after
emergence.
Inoculation treatments
Mycorrhizal inoculum was bulked in an open-pot culture of Zea mays L. and consisted
of soil, spores, mycelia and infected root fragments. The AM species used were three
strains isolated from Cabo de Gata Natural Park (Almería, Spain): Rhizophagus
intraradices (previously named Glomus intraradices), Septoglomus constrictum and
Claroideoglomus etunicatum. A Rhizophagus intraradices strain from our culture
collection was also used. Appropriate amounts of each inoculum containing about 700
infective propagules (according to the most probable number test), were added to the
corresponding pots at sowing time just below maize seeds. Non-mycorrhizal control
plants received the same amount of autoclaved mycorrhizal inocula together with a 10
ml aliquot of a filtrate (< 20 μm) of the AM inocula in order to provide a general
microbial population free of AM propagules.
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Growth Conditions
The experiment was carried out under glasshouse conditions with temperatures ranging
from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 45 days
prior to salinization to allow adequate plant growth and symbiotic establishment. Three
concentrations (0, 66, and 100 mM NaCl) of saline solution were reached in the soil
substrate by adding appropriate dilutions of a stock 2 M saline solution. The
concentration of NaCl in the soil was increased gradually on alternative days to avoid an
osmotic shock. It took 8 days, to reach the desired 66 and 100 mM NaCl levels. Plants
were maintained under these conditions for an additional 30 days.
Symbiotic development
The percentage of mycorrhizal root infection in maize plants was estimated by visual
observation of fungal colonization after clearing washed roots in 10% KOH and
staining with 0.05% trypan blue in lactic acid (v/v), as described Phillips and Hayman
(1970). The extent of mycorrhizal colonization was calculated according to the gridline
intersect method (Giovannetti and Mosse, 1980).
Biomass production
At harvest (75 days after planting), the shoot and root system were separated and the
shoot dry weight (SDW) and root dry weight (RDW) was measured after drying in a
forced hot-air oven at 70ºC for two days.
Proline content
Free proline was extracted from 0.5 g of fresh leaves and roots (Bligh and Dyer, 1959).
The methanolic phase was used for quantification of proline content. Proline was
estimated by spectrophotometric analysis at 530 nm of the ninhydrin reaction according
to Bates et al. (1973).
Na+ and K+ ions were extracted from 0.05 g of ground leaf and root dry material with 10
ml of deionized water. This extract was diluted, and for analysis, an ICP plasma
analyzer (IRIS Intrepid II XDL, Thermo Electron Corporation) was used. Extractions
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Chapter 4
were made from five different plants of each treatment. Mineral analyses were carried
out by the Analytical Service of the Centro de Edafología y Biología Aplicada del
Segura, CSIC, Murcia, Spain.
Chloride anions were determined from an aqueous extraction from 0.4 g of fresh
vegetal material with 10 ml of deionized water following the method of Cataldo et al.
(1975). The quantification of Cl- in roots and leaves was made from five different plants
of each treatment as described by Diatloff and Rengel (2001).
RNA was extracted using the RNeasy plant mini kit (Qiagen, Valencia, CA, U.S.A.)
from maize roots samples stored at -80ºC. Single-strand cDNA was primed by random
hexamers using 100-1,000 ng of DNase-treated RNA. RNA samples were denatured at
65ºC for 5 min and then reverse transcribed at 25ºC for 10 min and 42ºC for 50 min in a
final volume of 20 μl containing 10 μl of total RNA, 10 μM random primers
(Invitrogen, Carlsbad, CA, USA), 0.5 mM dNTPs, 10 U RNase inhibitor, 4 μl of 5x
buffer, 2 μl 0.1 M DTT, and 1 μl of Superscript II Reverse Transcriptase (Invitrogen).
The samples were precipitated with 1 (v/v) isopropanol and suspended in 20 μl of water.
Quantitative PCR
Gene expression analyses were carried out by quantitative reverse transcription (qRT)-
PCR using an iCycler iQ apparatus (BioRad, Hercules, CA, U.S.A.). The cDNA
samples were standardized to four reference genes: alpha tubulin (gi:450292),
elongation factor 1-alpha (EF1-α) (gi:2282583), polyubiquitin (gi:248338) and
glyceraldehyde phosphate dehydrogenase (GADPH) (gi:22237). The same reactions
were performed with specific primers designed for each of the analyzed genes:
ZmAKT2, For (5´-CCTCAAGCATCAGGTCGAGA-3´) and Rev (5´-
CTCTGTAATCTTCCTGGACG-3´), ZmSKOR, For (5´-
TCAGATCCAAGATGTCCCAG-3´) and Rev (5´-TTCGTATCCTCTTAACGCAG-3´)
ZmSOS1, For (5´-GCTTGTCACATACTTCACAG-3´) and Rev (5´-
ACTTGTCCACTTCACTACAC-3´). Individual real-time RT-PCR reactions were
assembled with oligonucleotide primers (0.15 μM each), 10.5 μl of 2x iQSYBR Green
Supermix (Bio-Rad; containing 100 mM KCl, 40 mM Tris-HCl pH 8.4, 0.4 Mm dNTPs,
50 U/μl iTaq DNA polymerase, 6 mM MgCl2, 20 nM SYBR Green I, 20 nM
fluorescein) plus 1 μl of a 1:10 dilution of each corresponding cDNA in a final volume
of 21 μl. Experiments were repeated three times, with the threshold cycle (CT)
determined in triplicate, using cDNAs that originated from three RNAs extracted from
three different biological samples.
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The relative levels of transcript were calculated using the Normalization Factor
(NF) based on the expression levels of the three best-performing housekeeping genes, in
our case polyubiquitin, GADPH and EF1-α. NF was measured using a Visual Basic
application for excel (GeNorm) that calculates the gene stability as described by
Vandesompele et al. (2002). The calculation was done for each cDNA used in the Q-
PCR quantification. Expression levels were transformed from Cq values using the PCR
efficiencies (Ramakers et al., 2003).
Statistical Analysis
Statistical analysis was performed using SPSS 19.0 statistical program (SPSS Inc.,
Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey test
with P< 0.05 as the significance cut-off. Two independent statistical analyses were
carried out: the first to analyze data from the different AMF treatments within each
saline level and the second one to analyze data from each fungal species at increasing
salinity.
Results
Symbiotic development
The increase of salt application affected negatively the shoot biomass production in all
treatments; although in the case of non-mycorrhizal plants the decrease was not
significant (Fig. 2A). In all AM treatments, the decrease was more evident at the highest
salt level applied (100 mM NaCl). In contrast, the root biomass production only
decreased with salinity in the non-mycorrhizal plants (Fig. 2B). When results were
analyzed for the three salt treatments it was clear that Ri CdG and Ce CdG, both
enhanced maize shoot biomass as compared to the non-mycorrhizal plants, while Ri
collect and Sc CdG did not (Fig. 2A).
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Fig. 1. Percentage of mycorrhizal root length in maize plants. Grey bars represent plants inoculated with
the collection Rhizophagus intraradices strain (Ri collect); white bars, plants inoculated with the native
Rh. intraradices CdG strain (Ri CdG); lined bars, plants inoculated with the native Septoglomus
claroideum CdG strain (Sc CdG) and dotted bars, plants inoculated with the native Claroideoglomus
etunicatum CdG strain (Ce CdG). Plants were subjected to 0, 66 or 100 mM NaCl. Different letters
indicate significant differences (p < 0.05) among fungal treatments at each salt level (a, b, c, d) or among
salt levels for each AMF treatment: Ri collect (D, E, F), Ri CdG (G, H, I), Sc CdG (J, K, L) or Ce CdG
(W, X, Y).
G W W
6
Shoot dry weight (g.plant -1)
a GH a X A
a H
5 A D J A a a
DE J A a
b b b b
4 b b b E K
c
c
3
NM
2 Ri collect
Ri CdG
1 Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM
Root dry weight (g.plant -1)
3 W B
W G W
2.5 A G a
D a a
a D ab J B D G a B
ab J ab J
2
b ab b ab ab
b b
1.5
1
0.5
0
0 mM 66 mM 100 mM
Fig. 2. Shoot (A) and root (B) dry weights (g plant-1) in maize plants. Black bars represent non-
mycorrhizal control plants (NM); grey bars, plants inoculated with the collection Rhizophagus
intraradices strain (Ri collect); white bars, plants inoculated with the native Rh. intraradices CdG strain
(Ri CdG); lined bars, plants inoculated with the native Septoglomus claroideum CdG strain (Sc CdG) and
dotted bars, plants inoculated with the native Claroideoglomus etunicatum CdG strain (Ce CdG). Plants
were subjected to 0, 66 or 100 mM NaCl. Different letters indicate significant differences (p < 0.05)
among fungal treatments at each salt level (a, b, c, d) or among salt levels for each AMF treatment: NM
plants (A, B, C), Ri collect (D, E, F), Ri CdG (G, H, I), Sc CdG (J, K, L) or Ce CdG (W, X, Y).
132
Chapter 4
Accumulation of proline
The accumulation of proline was more pronounced in root than in shoot tissues (Fig.
3A,B) and it increased in the roots with increasing salinity in the growth medium,
except for plants inoculated with Sc CdG, where the differences were not significant
(Fig. 3B). The highest accumulation of proline occurred in the roots of non-mycorrhizal
plants at 100 mM NaCl. It also increased with salinity in roots of plants inoculated with
Ri CdG and Ce CdG. In shoots, no significant differences were found either as a
consequence of increasing salinity or by the AM fungus inoculated.
W
0.6
A a A
0.5 A G
a D a D a
A a J
0.4 G W ab J W
a D J G a
0.3 a a
a
a ab ab
b NM
Proline content (μmol.g-1 DW)
0.2 Ri collect
Ri CdG
0.1 Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM
A
2 a B
D G
1.5 AB ab ab W
a DE J W bc
ab H J
1 B ab ab c
J b
a E
I a X
0.5 a
a a
0
0 mM 66 mM 100 mM
Fig. 3. Shoot (A) and root (B) proline accumulation in maize plants. See legend for Fig. 2.
Potassium
The increase of salinity in the growing medium decreased the accumulation of K+ in the
root tissues in all treatments, except in plants inoculated with Ri collect, which had
similar K+ levels at 0 mM NaCl and at 100 mM NaCl (Fig. 4B). In contrast, in shoot
tissues, maize accumulated more K+ at increasing salinity levels (Fig. 4A). This was
especially evident in plants inoculated with Sc CdG. At 100 mM NaCl, all the
mycorrhizal treatments accumulated more K+ in roots than the non-mycorrhizal plants
(Fig. 4B). At this salt level no significant differences in K+ accumulation were observed
133
Chapter 4
in roots between AM and non-AM treatments. In shoots, at all salinity levels, the non-
mycorrhizal plants and the plants inoculated with Ri collect exhibited always a lower K+
accumulation than plants inoculated with either of the three native AM fungal strains
(Ri CdG, Sc CdG or Ce CdG) (Fig. 4A).
J
J a
4
G a W G
W A
3.5 K ab b
H a X A DE
b b
3 a A D
a c c c
2.5 B E c
2 b b NM
Ri collect
1.5
Ri CdG
1 Sc CdG
0.5 Ce CdG
K+ content (%)
0
0 mM 66 mM 100 mM
2 B
G J
D
a aW
1.5 A a a DE H
H K X K X
a a a
B E ab ab a B a a
1 b ab b
0.5
0
0 mM 66 mM 100 mM
Fig. 4. Shoot (A) and root (B) potassium concentration in maize plants. See legend for Fig. 2.
Sodium
The accumulation of Na+ in maize plants increased considerably both in shoot and in
root tissues when the plants were cultivated under salinity (Fig. 5 A,B). When data were
analyzed within each salt level, it was observed that at 0 mM NaCl the three native
AMF enhanced the accumulation of Na+ in root tissues as compared to the non-
mycorrhizal plants or those inoculated with Ri collect (Fig. 5B). However, at 66 mM
NaCl and 100 mM NaCl no significant differences in Na accumulation in roots were
observed among treatments, except that plants inoculated with Ce CdG, reduced
significantly this parameter at 100 mM NaCl. In the shoot tissues, it was observed that
at 0 mM NaCl the levels of Na+ were very low in all treatments (Fig. 5A). The
accumulation of Na+ was enhanced at 66 and 100 mM NaCl for all treatments, with
non-mycorrhizal plants exhibiting the highest Na+ accumulation and mycorrhizal plants
the lowest, especially those inoculated with Ce CdG (Fig. 5A).
134
Chapter 4
2.5 A
A
a
2
D J
B
1.5 b G b
a bc
E W NM
H K Ri collect
1 b c
b b X Ri CdG
b
Na+ content (%)
0.5 C F I L Y Sc CdG
a a a a a Ce CdG
0
0 mM 66 mM 100 mM
A J
3.5
B G a DG a B
3 E a ab
ab a K W W
ab ab b
2.5 b
2
H L X
1.5
ab b a
1 C F
0.5 c c
0
0 mM 66 mM 100 mM
Fig. 5. Shoot (A) and root (B) sodium concentration in maize plants. See legend for Fig. 2.
A
16 G J
B H K a D A
ab W
14 E bc b
a ab a X c
12 bc
I L c
Y
10 C F b a
c NM
8 d cd
Ri collect
6
Ri CdG
Cl- content (mmol.g-1 DW)
4 Sc CdG
2 Ce CdG
0
0 mM 66 mM 100 mM
J
J A
30 D a
B G a W a D G W B
J a a a
25 a a a a
a
20 H X
C E
ab ab
15 b ab
10
5
0
0 mM 66 mM 100 mM
Fig. 6. Shoot (A) and root (B) chloride concentration in maize plants. See legend for Fig. 2.
135
Chapter 4
Chloride
The accumulation of Cl- increased in both shoot and root tissues with increasing salinity
in the growth medium (Fig. 6 A,B). This was more evident in the shoot tissues (Fig.
6A). When data were analyzed within each salt level, it was observed that in roots no
remarkable differences in Cl- accumulation were found among treatments. In shoots, at
0 mM NaCl, plants inoculated with the three native AMF (Ri CdG, Sc CdG and Ce
CdG) accumulated more Cl- than non-mycorrhizal plants or those inoculated with the Ri
collect fungus (Fig. 6A). In contrast, when salt was applied to the growth medium, non-
mycorrhizal plants always exhibited the highest Cl- accumulation and no important
differences in Cl- accumulation were observed among fungal treatments. At both saline
levels, plants inoculated with Ce CdG showed the lowest accumulation of Cl-.
J
D
400 a
a G A
350 ab
300 A
bc NM
250 W
c Ri collect
200 Ri CdG
Sc CdG
150
Ce CdG
100
15
X
a
10 K
H X
b
E bc K a
5
B
cd E H
B b
d cd bc
d
K+/Na+ ratio
0
0 mM 66 mM 100 mM
A D
4 a a B
3.5
3
2.5
G J
2
b b W
1.5 b
K X H X
1 B E H B E ab K
ab a a
0.5 c bc bc c ab bc
0
0 mM 66 mM 100 mM
Fig. 7. Shoot (A) and root (B) K+/Na+ ratio in maize plants. See legend for Fig. 2.
136
Chapter 4
K+/Na+ ratios
The K+/Na+ ratio was negatively affected by salinity in both shoots and roots (Fig.
7A,B). However, the effect was more evident in shoot tissues, where the differences
between the non-saline treatment and either of the two saline treatments were of two
orders of magnitude (Fig. 7A). In roots, the K+/Na+ ratio at 0 mM NaCl was lower in
plants inoculated with either of the three native AMF as compared to non-mycorrhizal
plants or plants inoculated with Ri collect (Fig. 7B). In contrast, when salt was applied
non-mycorrhizal plants showed the lowest K+/Na+ ratio, especially if compared to roots
of plants inoculated with Ce CdG. In the shoots, at both saline levels, the lowest K+/Na+
ratios were also found in non-mycorrhizal plants and in plants colonized with the Ri
collect strain (Fig. 7A). The three native AMF (Ri CdG, Sc CdG and Ce CdG) showed
significantly enhanced K+/Na+ ratios in shoots as compared to the non-mycorrhizal
plants, especially those colonized by Ce CdG.
Ion analyses suggest that AMF affect tissue K+ and Na+. We therefore tested whether
membrane transporters involved in shoot K+ and Na+ deposition were affected at the
transcript level by AMF colonization.
The expression of the ZmAKT2 gene was differently affected by increasing
salinity in the different fungal treatments (Fig. 8A). In fact, in roots of non-mycorrhizal
plants or plants colonized by the Ri collect strain, it decreased its expression at 66 and
100 mM NaCl as compared to 0 mM NaCl. In contrast, the expression of this gene
increased steadily with increasing salinity in roots of plants colonized by Sc CdG and
Ce CdG. When data were analyzed within each salt level, it was observed that in the
absence of salt in the growth medium the expression of ZmAKT2 was notably higher in
roots of non-mycorrhizal plants and plants colonized by the Ri collect strain than in
roots of plants colonized by either of the three native AM fungal strains. At 66 mM
NaCl, few differences among treatments were observed. Finally, at the highest salt level
(100 mM NaCl), the expression of this gene increased notably in roots of plants
colonized by Sc CdG and Ce CdG, as compared to the other treatments.
The expression of the ZmSOS1 gene was negatively affected by the highest
salinity level in non-mycorrhizal plants or plants inoculated with Ri collect and Ri CdG
(Fig. 8B). In contrast, the application of 100 mM NaCl enhanced considerably the
expression of the ZmSOS1 gene in roots of plants colonized by Ce CdG as compared to
66 mM NaCl or to the non-saline level. When data were analyzed within each salt level,
it was observed that in the absence of salinity plants inoculated with Sc CdG or with Ce
CdG had a significantly lower expression of ZmSOS1 than the non-mycorrhizal plants.
No significant differences among treatments were observed at 66 mM NaCl, while at
137
Chapter 4
the highest salt level (100 mM NaCl) plants inoculated with Ce CdG exhibited the
highest expression of this gene in their roots.
The expression of the ZmSKOR gene in roots was little affected by the
increasing salinity in non-mycorrhizal plants or in plants inoculated with the Ri collect
and Ri CdG strains (Fig. 8C). In contrast, in plants colonized by Sc CdG and Ce CdG,
the expression of this gene increased steadily with increasing salinity. When data were
analyzed within each salt level, it was observed that in the absence of salinity plants
inoculated with Sc CdG or with Ce CdG had a significantly lower expression of
ZmSKOR than all other treatments. This lower expression was maintained at 66 mM
NaCl in roots of plants colonized by Ce CdG, but again, at the highest salt level (100
mM NaCl) plants inoculated with Ce CdG and also plants inoculated with Sc CdG
exhibited the highest expression of this gene.
W
ZmAKT2 relative expression
2 a
J A
a
1.5
A D
a a
1 B
K NM
G b Ri collect
a X G
0.5 b L X C H E b Ri CdG
ab Sc CdG
b b b b c Ce CdG
0
0 mM 66 mM 100 mM
ZmSOS1 relative expression
1 W
A a B
D
0.8 a D
ab G a G
J
ab X AB a a X B E JK
0.6
a a bc bc b
K b
0.4 c H
c
0.2
0
0 mM 66 mM 100 mM
ZmSKOR relative expression
2 C
J
1.5
a W
G A D
1 a a a K a
A DE A H
a
a a b E b
X H X
0.5 L b
b b b
b
0
0 mM 66 mM 100 mM
Fig. 8. Analysis of ZmAKT2 (A), ZmSOS1 (B) and ZmSKOR (C) genes expression by real time quantitative
RT-PCR in roots of maize plants from the different treatments. See legend for Fig. 2.
138
Chapter 4
Discussion
Previous studies have demonstrated that maize plants inoculated with AMF grow better
than non-mycorrhizal plants under salt stress conditions (Feng et al., 2002; Sheng et al.,
2008; Sheng et al., 2011). However these experiments were based on the inoculation of
a sole AM fungus, Glomus mosseae. Although mycorrhizal symbiosis is usually
considered nonspecific, Klironomos (2003) found that there was great variation in
growth performance with the same fungal isolate. Therefore, AM colonization would
depend on the compatibility of both AMF and host plants. In the present work, we
assessed the performance of three AM fungal species isolated from a saline area and
compared their effectiveness with a G. intraradices (= Rhizophagus intraradices) strain
belonging to the Zaidin Experimental Station collection. Each species exhibited
different mycorrhizal development and symbiotic efficiency under the salt levels
assayed.
The results of the present study showed that colonization rates varied among
fungal species. Ri collect had the higher rate of root colonization, followed by Ce CdG,
Sc CdG and Ri CdG. While most studies reported a decrease in mycorrhizal
colonization at increasing salinity levels (Sharifi et al., 2007; Evelin et al., 2012), our
study reveals a significant increase in root colonization in three of the AMF strains, Ri
collect, Sc CdG and Ce CdG, while Ri CdG had the same colonization rate at all salinity
levels. Yamato et al. (2008) also found that colonization rates were not reduced in any
of the AMF present in coastal vegetation on Okinawa Island. Similar results were
reported by Wu et al. (2010) in citrus colonized by Paraglomus occultum isolated from
a saline habitat. These results may be explained in terms of salt-tolerance of the AMF
isolates: they can maintain or even increase colonization capacity under saline
conditions. On the other hand, Ri collect has been previously described to have a very
high rate of colonization (Graham et al., 1996; Ruiz-Lozano et al., 2001), thus it seems
not surprising that it maintained or even increased the colonization rate. Nevertheless,
under saline conditions the native AMF strains isolated from saline areas maintained a
higher symbiotic efficiency with maize plants than the collection strain.
Plant biomass production is an integrative measurement of plant performance
under many types of abiotic stress conditions and the symbiotic efficiency of AMF has
been measured in terms of plant growth improvement (see reviews by Evelin et al.,
2009; Ruiz-Lozano et al., 2012). In our experiment, maize plants inoculated with Ri
CdG and Ce CdG had the highest shoot dry biomass at all salinity levels, demonstrating
the higher symbiotic efficiency of these native AMF (Oliveira et al., 2005; Querejeta et
al., 2006). The growth of maize inoculated with Gi collect was similar to the non-
mycorrhizal plants, except at 100 mM NaCl, where it was lower. The latter can be
explained due to the high percentage of root colonization by this fungal strain that could
demand excessive carbohydrates from the plant. In fact plant growth responses to AMF
inoculation can range from parasitic to mutualistic (Klironomos, 2003). Sc CdG had a
139
Chapter 4
140
Chapter 4
2009; Talaat and Shawky, 2011; Evelin et al., 2012). Results are also consistent with
Giri et al. (2007), who showed higher accumulation of K+ by mycorrhizal plants in
saline soils, thus maintaining a high K+/Na+ ratio which influences the ionic balance of
the cytoplasm or Na+ efflux from plants. Recently Hammer et al, (2011) demonstrated
that Rh. intraradices can selectively take up elements such as K+, Mg2+ and Ca2+ while
avoiding Na+ uptake. The latter indicates that the AM fungal mycelium might pre-select
nutrients for the plants. Moreover, as a significant proportion of elemental nutrient
uptake in plants occurs via mycorrhizal fungi, they help to alleviate the effects of the
excess of salts in the soil. Our results confirm that Se. constrictum also induced a higher
K+/Na+ ratio compared to non-mycorrhizal plants. In the roots, levels of Na+ were
always higher than in the leaves. It has been proposed that in AM-inoculated pants, Na+
might be kept inside root cell vacuoles and intraradical fungal hyphae to prevent the
allocation of Na+ to the shoots (Cantrell and Linderman, 2001). Plants inoculated with
Ce CdG had the lowest Na+ concentration at 66 and 100 mM of NaCl, being the most
efficient fungus in terms of avoiding Na+ uptake. Hammer et al. (2011) found different
concentrations and distributions of Na+ and Cl- within the fungal tissue and they
hypothesized that AMF exclude Na+ but include Cl-. Our results showed that all
treatments enhanced Cl- concentration as salinity in the growing medium increased.
Mardukhi et al. (2011) proposed that mycorrhizal plants had no control on plant Cl-
uptake. Thus the alleviating effect of AMF on plant growth under salinity stress is more
related to Na+ than Cl- uptake. Moreover, for maize, Na+ causes higher ion toxicity than
Cl- (Fortmeier and Schubert, 1995).
The previous results prompted us to hypothesize that AMF may have regulated
the expression of plant genes encoding for ion transporters. It is well documented that
overexpression of Na+/H+ and K+/H+ antiporters improve salt tolerance in plants (Zhang
et al., 2001; Rodriguez-Rosales et al., 2008). However, scarce information is available
on the possible regulation by the AM symbiosis of plant genes involved in ion
homeostasis. Until now, only Ouziad et al. (2006) have studied the effect of AM
symbiosis on the expression of two Na+/H+ antiporters in tomato under salt stress
conditions, showing no regulation of these genes by the AM symbiosis. Nevertheless,
we studied the expression of three genes involved in Na+ and K+ transport in order to
get some clues on molecular mechanisms involved in the enhanced tolerance of
mycorrhizal plants to salinity stress.
The SOS signalling pathway has been pointed out to have a major role
maintaining ion homeostasis by regulating Na+ and K+ transport at both the plasma
membrane and tonoplast (Zhu, 2002, 2003). AKT family contributes to a major
potassium acquisition by plants and SKOR transports K+ to the shoots (Munns, 2005).
Based on that, we analyzed the expression of some of these maize genes in the roots of
the different treatments. The most important differences among treatments were
observed at 100 mM NaCl for the three genes, where plants inoculated with Se CdG or
with Ce CdG exhibited enhanced relative expression. In contrast, under non saline
141
Chapter 4
Acknowledgements
This work was financed by two research projects supported by Junta de Andalucía
(Spain). Projects P06-CVI-01876 and P11-CVI-7107. We thank Sonia Molina for
technical assistance and Domingo Álvarez (curator of the EEZ germplasm collection),
for taking care of the native AMF inocula. We also thank Dr. Jean Charles Isner for
essential support in primer design.
142
Chapter 4
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CHAPTER 5
Resumen
La alta salinidad del suelo es un serio problema para la producción agrícola porque la
mayoría de las plantas cultivadas son sensibles sal. Este es el caso de un cultivo tan
importante como el maíz. El estrés salino conduce a un estrés oxidativo secundario en
plantas y se ha demostrado en varias especies de plantas una correlación entre la
capacidad antioxidante y tolerancia a la salinidad. La capacidad antioxidante de la
planta puede ser potenciada por los hongos micorrícicos arbusculares (MA) y se ha
propuesto que la simbiosis MA es más eficaz con especies nativas que con exóticas. Por
lo tanto, se investigó si hongos nativos MA aislados de un ambiente salino pueden
ayudar a las plantas de maíz a superar el estrés salino mejor que hongos MA de
colección y si la protección frente al estrés oxidativo está implicada en este efecto. Las
plantas de maíz inoculadas con los tres hongos nativos MA mostraron una mayor
eficiencia del fotosistema II y la conductancia estomática, lo que sin duda contribuyó a
reducir la fotorrespiración y la producción de ROS. De hecho, la acumulación de
peróxido de hidrógeno, el daño oxidativo a lípidos y la pérdida de electrolitos en la
membrana de estas plantas MA fueron significativamente más bajos que en las no
micorrizadas o en plantas inoculadas con el hongo MA de colección. La activación de
enzimas antioxidantes como la superóxido dismutasa o catalasa también contribuyeron
a estos efectos.
151
152
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Beatriz Estrada, Ricardo Aroca, José Miguel Barea and Juan Manuel Ruiz-
Lozano*
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Abstract
High soil salinity is a serious problem for crop production because most of the
cultivated plants are salt sensitive. This is the case of a so important crop such as maize.
Salinity stress leads to secondary oxidative stress in plants and a correlation between
antioxidant capacity and salt tolerance has been demonstrated in several plant species.
The plant antioxidant capacity may be enhanced by arbuscular mycorrhizal fungi
(AMF) and it has been proposed that AM symbiosis is more effective with native than
with exotic AMF species. Thus, we investigated whether native AMF isolated from a
saline environment can help maize plants to overcome salt stress better than AMF from
collection and whether protection against oxidative stress is involved in such an effect.
Maize plants inoculated with three native AMF showed higher efficiency of
photosystem II and stomatal conductance, which surely contributed to decrease
photorespiration and ROS production. Indeed, the accumulation of hydrogen peroxide,
the oxidative damage to lipids and the membrane electrolyte leakage in these AM plants
were significantly lower than in non-mycorrhizal plants or in plants inoculated with the
collection AMF. The activation of antioxidant enzymes such as superoxide dismutase or
catalase also accounted for these effects.
Key words: antioxidant system, arbuscular mycorrhiza, plant tolerance, salinity, salt-
adapted
1. Introduction
Maize (Zea mays L.) is one of the most important crops both for human and
animal consumption. This crop is cultivated on more than 142 million ha of land
worldwide and it is estimated to produce around 913 million tonnes of grain in the
period 2012/2013 [1], accounting for one third of the total global grain production [2].
In a climate change scenario, one expected threat is the increase in land salinization.
Over 6% of the world’s land is affected by salinity and its extent is increasing regularly
throughout the world [3], causing global agricultural losses equivalent to an estimated
US 12 billion a year [4]. Although salinity in soils may occur naturally, inappropriate
cultivation practices are also contributing to the salinization of the rhizosphere [5].
Nowadays, high salts in the soils are a very serious problem for crop production because
most of the cultivated plants are sensitive to salt stress (glycophytes) [6]. This is the
case of maize, which is particularly vulnerable to salinity [7]. Thus land salinization is a
major global issue because of its adverse impact on agricultural productivity,
sustainability and as a threat for food supply [8]. In arid and semi-arid regions, the issue
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is aggravated due to poor soil management practices together with limited rainfall, high
evapotranspiration, and high temperature rates [9].
Salinity stress leads to secondary oxidative stress in plants [10]. The latter is
produced when pathways are uncoupled in the metabolism of plants and electrons, with
high-energy state, are transferred to molecular oxygen to form reactive oxygen species
(ROS) [10]. ROS are normally produced at a low level in organelles such as
chloroplasts, mitochondria and peroxisomes. However, under salt-stress conditions,
their production dramatically increases due to the accumulation of NADPH and ATP
that cannot be consumed [11]. This modifies the balance between the formation and
removal of ROS species [12]. Singlet oxygen (1O2), superoxide radical (O2•−), hydroxyl
radical (OH•) and hydrogen peroxide (H2O2) can seriously disrupt normal metabolism
through denaturalization of proteins, mutation of DNA and lipid peroxidation [13].
Thus, plants need to have appropriate detoxification systems to allow rapid removal of
these compounds. They constantly sense the level of ROS and reprogramme their gene
expression to respond to changes in their environment [13]. The most common
mechanism to detoxify ROS produced during salt-stress response is the induction of
ROS-scavenging enzymes, such as superoxide dismutase (SOD) and catalase (CAT)
[14]. SOD converts O2•− to H2O2 and then CAT converts H2O2 to water and molecular
oxygen in peroxisomes. A correlation between antioxidant capacity and NaCl tolerance
has been demonstrated in several plant species [15]. Despite the several mechanisms
developed by plants to detoxify the excess of ROS production, most of the cultivated
plants are glycophytes and under high salts in the soil they cannot cope with the extra
production of ROS [6].
Fortunately, a number of beneficial soil microorganisms, particularly arbuscular
mycorrhiza fungi (AMF), help plants to cope with abiotic stress conditions [16]. AMF,
belonging to the phylum Glomeromycota, are considered the oldest group of organisms
living in symbiosis with terrestrial plants [17]. They are fundamental for soil fertility,
both in natural and agricultural ecosystems and most land plants are colonized by these
fungi [18]. AMF are widely found in saline soils [19-20]. In fact, several authors have
demonstrated the beneficial effect of AMF in growth and productivity under salt stress
conditions on glycophytes and halophytes, decreasing plant yield loses (reviewed by
[11, 21]. Among other mechanisms, some studies have demonstrated that AM
symbiosis enhances the activities of antioxidant enzymes, helping plants to alleviate salt
stress [22-23]. Most studies have focused in the use of collection species as AMF
inoculum [21]. However several studies showed that native AMF can perform better in
soils from which they are isolated: agricultural systems [24], polluted soils [25] and
semiarid degraded soils [26] among others. According to this, some authors reported
that AM symbiosis, is more effective with native than with exotic AMF species [27-29].
However, the mechanisms that allow AM plants to have higher salinity tolerance are far
from being understood [11]. The isolation and study of AMF isolated from a saline area
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will contribute to elucidate the ecophysiology of AMF under such stress conditions
[25].
The aim of the present study was to investigate whether native AMF isolated
from a saline environment can help maize plants to overcome the negative effects of salt
stress better than AMF from collection and whether protection against oxidative stress
is involved in such an effect. Plant physiological and biochemical parameters related to
the oxidative status were determined in non-AM maize plants and in maize plants
inoculated with different AMF isolates after a salt stress period.
2.1. Identification of the mycorrhizal strains isolated from Cabo de Gata Natural Park
AM fungal spores were separated from the soil samples by a wet sieving process
[30]. The morphological spore characteristics and their subcellular structures were
described from a specimen mounted in: polyvinyl alcohol-lactic acid-glycerine (PVLG)
[31]; a mixture of PVLG and Melzer’s reagent [32]; a mixture of lactic acid to water at
1:1; Melzer’s reagent; and water [33]. For identification of the AMF species, spores
were then examined using a compound microscope at up to 400-fold magnification as
described for glomeromycotean classification by Oehl et al. [34]. The species were
identified based on its spore morphology as a Rhizophagus intraradices [35],
Claroideoglomus etunicatum [36] and Septoglomus constrictum [37].
In addition to the morphological identification, a molecular identification was
also carried out. For that, spores isolated from the bait cultures of each fungal strain
were surface-sterilized with chloramine T (2%) and streptomycin (0.02%) and crushed
with a sterile disposable micropestle in 40 μL milli-Q water [38]. A two-step PCR was
conducted to amplify the AM fungal DNA from the spores. The first PCR step was
performed with the universal eukaryote primers NS1 and NS4 region of the small
subunit ribosomal gene and the second with the specific AM fungal primers AML1 and
AML2 [39]. The amplified DNA was purified using the Ilustra™ GFX™ PCR DNA
and Gel Band Purification Kit (GE Helthcare, UK). DNA fragments were sequenced on
an automated DNA sequencer (Perkin-Elmer ABI Prism 373). Sequence data were
compared to gene libraries (EMBL and GenBank) using BLAST program [40].
The BLAST analysis unambiguously placed Rhizophagus intraradices as the
closest relative of our Rhizophagus intraradices CdG strain, with sequence accession
number FR750209 [41] having a 99% identity. Septoglomus constrictum was the closest
relative to our Se. constrictum CdG strain, with sequence accession number FR750212
[41] having a 99% identity. Finally, Claroideoglomus etunicatum was the closest
relative of our Cl. etunicatum CdG strain, with sequence accession number FR750216.1
[41] having also a 99% identity. The AM fungal strains have been incorporated to the
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The experiment was carried out under glasshouse conditions with temperatures
ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 45 days
prior to salinization to allow adequate plant growth and symbiotic establishment. Three
concentrations (0, 66, and 100 mM NaCl) of saline solution were reached in the soil
substrate by adding appropriate dilutions of a stock 2 M saline solution. The
concentration of NaCl in the soil was increased gradually on alternative days to avoid an
osmotic shock. It took 8 days, to reach the desired 66 and 100 mM NaCl levels. Plants
were maintained under these conditions for additional 30 days.
At harvest (75 days after planting), the shoot and root system were separated and the
shoot dry weight was measured after drying in a forced hot-air oven at 70ºC for two
days.
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Stomatal conductance was measured two hours after light turned on by using a
porometer system (Porometer AP4, Delta-T Devices Ltd, Cambridge, UK) following
the user manual instructions. Stomatal conductance measurements were taken in the
second youngest leaf from five different plants from each treatment.
The electrolyte leakage was calculated on the third leaf of each maize plant from
a leaf sample of 3 x 1.5 cm as described by Verslues et al. [45]. The initial conductivity
(C0) was measured with a conductivity metre COND 510 (XS Instruments; OptoLab,
Milan, Italy) after subjecting the samples to incubation at 25ºC in 10 ml de-ionized
water overnight with continuous shaking at 100 rpm. The samples were then autoclaved
at 121ºC for 20 min. Final conductivity (CF) was measured after the samples had cooled
down to room temperature. The conductivity of distilled water was also measured and
referred as Cw. The percentage of electrolyte leakage was calculated as follows:
[(C0 – Cw) / (CF – Cw)] x 100.
Lipid peroxides were extracted by grinding 500 mg of leaves and roots with an
ice-cold mortar and 6 ml of 100 mM potassium phosphate buffer (pH 7). Homogenates
were filtered through one Miracloth layer and centrifuged at 15,000 × g for 20 min. The
chromogen was formed by mixing 200 μl of supernatants with 1 ml of a reaction
mixture containing 15% (w/v), trichloroacetic acid (TCA), 0.375% (w/v) 2-
thiobarbituric acid (TBA), 0.1% (w/v) butyl hydroxytoluene, 0.25N HCl and then
incubating the mixture at 100ºC for 30 min [46]. After cooling at room temperature,
tubes were centrifuged at 800 × g for 5 min and the supernatant was used for
spectrophotometric reading at 532 nm. Lipid peroxidation was estimated as the content
of 2-thiobarbituric acid-reactive substances (TBARS) and expressed as equivalents of
malondialdehyde (MDA) according to Halliwell and Gutteridge [47]. The calibration
curve was made using MDA in the range of 0.1-10 nmol. A blank for all samples was
prepared by replacing the sample with extraction medium, and controls for each sample
were prepared by replacing TBA with 0.25N HCl. In all cases, 0.1% (w/v) butyl
hydroxytoluene was included in the reaction mixtures to prevent artifactual formation of
TBARS during the acid-heating step of the assay.
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Enzyme extraction was done as described before by Aroca et al. [50] with slight
modifications. Five hundred mg of leaf and root fresh tissues were homogenized
separately in a cold mortar with 4 ml of 100 mM phosphate buffer (pH 7.0) containing
0.1 mM DTPA (diethylenetriamine pentaacetic acid; a metal chelating agent) and 40 mg
PVPP (polyvinylpolypyrrolidone), which removes phenolics and alkaloids from plant
extracts, avoiding interference with spectrophotometric measurements and enhancing
enzyme stability. The homogenate was centrifuged at 20,000 x g for 20 min at 4ºC. The
supernatant was separated and used to determine the activity of antioxidant enzymes.
All enzymatic activities were measured in a spectrophotometer InfiniteR 200 PRO series
(Tecan Trading AG, Switzerland) at 25 °C. Total protein was assayed by the method
described by Bradford [51].
Total superoxide dismutase (SOD) (EC 1.15.1.1) activity was measured
according to Becana et al. [52]. One unit of SOD activity (U) was defined as the amount
of enzyme which produced a 50% inhibition of nitroblue tetrazolium (NBT) reduction.
The reaction mixture (1,9 ml) contained 50 mM phosphate buffer (pH 7.8), 14,3 M
methionine, 82,5 μM NBT, 2,2 μM riboflavin and 100 μl enzyme extract. Riboflavin
was added last and tubes were shaken and illuminated with fluorescent light. The
reaction was allowed to proceed for 10 min until the colour of the blank shifted to dark
violet. Absorbance of the reaction mixture was read at 560 nm.
Catalase (CAT) (EC1.11.1.6) activity was assayed as described by [53].
Consumption of H2O2 (extinction coefficient of 39.6 mM-1cm-1) at 240 nm for 1 min
was monitored. The reaction mixture consisted of 50 mM phosphate buffer (pH 7.0)
containing 10 mM H2O2 and 5 μl of enzyme extract in a 205 μl volume.
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Statistical analysis was performed using SPSS 19.0 statistical program (SPSS
Inc., Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey
test with P< 0.05 as the significance cut-off. Two independent statistical analyses were
carried out: the first to analyze data from the different AMF treatments within each
saline level and the second one to analyze data from each fungal treatment at increasing
salinity.
3. Results
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CdG (Fig. 1B). When plants were subjected to 100 mM NaCl all the mycorrhizal plants
showed higher photosynthetic efficiency than non-mycorrhizal plants, mainly plants
inoculated with Ri CdG and Ce CdG (Fig. 1B).
0 0 4.03 ± 0.14 Ab
NM 66 0 3.95 ± 0.12 Ab
100 0 3.69 ± 0.11 Ab
Table 1. Percentage of mycorrhizal root colonization and shoot dry weight (g plant-1) in maize plants.
NM represents non-mycorrhizal control plants; Ri collect, plants inoculated with the collection
Rhizophagus intraradices strain; Ri CdG, plants inoculated with the native Rh. intraradices CdG strain;
Sc CdG, plants inoculated with the native Septoglomus claroideum CdG strain and Ce CdG, plants
inoculated with the native Claroideoglomus etunicatum CdG strain. Plants were subjected to 0, 66 or 100
mM NaCl. Different letters indicate significant differences (p < 0.05) among fungal treatments at each
salt level (a, b, c, d) or among salt levels for each AMF treatment: NM plants (A, B, C), Ri collect (D, E,
F), Ri CdG (G, H, I), Sc CdG (J, K, L) or Ce CdG (W, X, Y).
The application of 66 or 100 mM NaCl did not significantly affect the electrolyte
leakage of maize plants as compared to plants growing in the absence of salinity (Fig.
1C). However, at each saline level non-mycorrhizal plants had always the highest values
of electrolyte leakage, followed by plants inoculated with Ri collect. In contrast, maize
plants inoculated with any of the three native AMF always showed significantly lower
electrolyte leakage than control non-mycorrhizal plants (Fig. 1C).
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35 J
Stomatal conductance
A G a W A
(mmolH2Om-2s-1)
30 a D a a
25 a X X
H K a
H
20 B E a ab K a
b b B a NM
a
15 b F Ri collect
Ri CdG
10 c Sc CdG
Ce CdG
5
0 mM 66 mM 100 mM
G W
0.7
Photosynthetic efficiency
J G B
a a X
A D a
0.6 A D a JK H X
b b b
b b b a K a
E ab
0.5
b
B
0.4 c
0.3
0.2
0 mM 66 mM 100 mM
25
C
(%)
A
leakage(%)
A A D a
20 D
ElectrolyteLeakage
a D G J W a ab J W W
a G bc c b G J bc
15 b b b c c
c
Electrolyte
10
0
0 mM 66 mM 100 mM
Fig. 1. Stomatal conductance (mmol H2O m-2s-1) (A), efficiency of photosystem II (B) and electrolyte
leakage (C) in maize plants. Black bars represent non-mycorrhizal control plants (NM); grey bars, plants
inoculated with the collection Rhizophagus intraradices strain (Ri collect); white bars, plants inoculated
with the native Rh. intraradices CdG strain (Ri CdG); lined bars, plants inoculated with the native
Septoglomus claroideum CdG strain (Sc CdG) and dotted bars, plants inoculated with the native
Claroideoglomus etunicatum CdG strain (Ce CdG). Plants were subjected to 0, 66 or 100 mM NaCl.
Different letters indicate significant differences (p < 0.05) among fungal treatments at each salt level (a, b,
c, d) or among salt levels for each AMF treatment: NM plants (A, B, C), Ri collect (D, E, F), Ri CdG (G,
H, I), Sc CdG (J, K, L) or Ce CdG (W, X, Y).
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Ri CdG
5
(nmol MDA g-1 DW)
Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM
25 F
D B
20 A a
E a
a J W
15 F
D B WX G J b
B
a G b G b b
10 a X b b
b
b K b
5 b
0
0 mM 66 mM 100 mM
Fig. 2. Shoot (A) and root (B) oxidative damage to lipids in maize plants. See legend for Fig. 1.
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Cabo de Gata than in the non-mycorrhizal plants or plants colonized by Ri collect (at
100 mM NaCl) (Fig. 3B).
0.8 A
A D
0.7
a a J
0.6
E G
J B E G J G ab W
0.5 W
a W a a a a b ab
Hydrogen peroxide (μmol g-1 DW)
0.4 B ab ab ab
a
b NM
0.3 Ri collect
0.2 Ri CdG
Sc CdG
0.1 Ce CdG
0
0 mM 66 mM 100 mM
A
0.9 a B
0.8 A
0.7 D
a
0.6 D a
0.5 W
J W
ab
G J b G J W
0.4 B E
ab a b b b b b
0.3 ab ab H
0.2 b
0.1
0.0
0
0 mM 66 mM 100 mM
Fig. 3. Shoot (A) and root (B) hydroxide peroxide content in maize plants. See legend for Fig. 1.
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Chapter 5
J W
12 A
a D a
A J A J
10 a
DE X a a WX a E G a
8 ab GH a a
H b a
a
6
SOD activity (U min-1mg-1Prot)
bc NM
4 B Ri collect
Ri CdG
2 c Sc CdG
Ce CdG
0
0 mM 66 mM 100 mM
W
25 a B
X G J
20 J
a b b
D A D a
15 H J Y
A a a a a ab ab A
H D
a c
10 b c
0
0 mM 66 mM 100 mM
Fig. 4. Shoot (A) and root (B) SOD activity in maize plants. See legend for Fig. 1.
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D
18 a A
16 A
14 W
ab G a
J W
CAT activity (nmol H2O2 min-1mg-1Prot)
12
GH B E a
10 J cd bc J NM
8 B H a X d b b
b Ri collect
6 a F a a Ri CdG
4 a Sc CdG
2 Ce CdG
0
0 mM 66 mM 100 mM
18 D
a G W B
16
14 a a
G J A
J
12 a a a
E X a
10
b b
8
B
6 B
F H K Y c
4 a
a a a a
2
0
0 mM 66 mM 100 mM
Fig. 5. Shoot (A) and root (B) CAT activity in maize plants. See legend for Fig. 1.
4. Discussion
Excessive soil salinity is well known to induce oxidative stress in plants [54].
Previous studies have suggested that tolerance of plants to salt stress is associated with
the induction of antioxidant enzymes and reduction of oxidative damage [55-56]. AM
symbiosis enhances the activity of antioxidant enzymes in order to help plants to cope
with the reactive oxygen species generated by salinity [22-23, 57]. Nevertheless the
response of the individual enzymes varies with respect to the host plant and the fungal
species involved in the association [11]. Results from the present work confirms that
native AMF reduced the oxidative damage in the host plant, but also that they differed
in their response to salinity, in the modulation of the antioxidative capacity of the host
plant and in the promotion of plant growth under saline conditions. The degree of root
colonization also varied among fungal species, but the literature reflects that there is no
threshold value of root colonization for enhancement of plant fitness and this depends
on the plant and the fungal species involved [58]. In fact Ri collect had higher
colonization rate than the autochthonous AMF but it did not show better symbiotic
efficiency.
In this study, AM plants may improve the net assimilation rates by protecting
photochemical processes of photosystem II and enhancing stomatal conductance when
subjected to salinity stress. Plants inoculated with native AMF exhibited better
performance of photosystem II and higher stomatal conductance when subjected to high
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salinity level (100 mM NaCl). These results indicate less damage in the photosynthetic
machinery and enhanced transpiration rates in maize plants inoculated with native
AMF. Sheng et al. [59] and Hajiboland et al. [60] reported a similar tendency, and
Querejeta et al. [27] showed that the enhancement of plant stomatal conductance was
higher with native AMF than with collection fungi. These two effects may have
accounted for the enhanced tolerance of AM plants, particularly native inoculated ones,
most likely by enhancing CO2 fixation and plant growth during salinity stress. In any
case, the native Sc CdG also improved these physiological parameters but it did not
enhance plant growth, demonstrating a differential ability by each fungus or that an
extended growing period was needed to achieve a positive effect on plant growth.
The above mentioned processes could have also contributed to decrease
photorespiration and then lead to lower ROS production in AM plants [10]. Indeed, the
accumulation of hydrogen peroxide under salinity was considerably lower in roots of
plants inoculated with the three native AMF and the oxidative damage to lipids was also
lower in shoots and roots of these AM plants. Several studies have reported lower H2O2
accumulation in AM plants [60-61]. Our results demonstrate that colonization of maize
plants by salinity-adapted AMF lead to a lower accumulation of ROS species. Salt
stress injuries the membrane integrity, which produces ion leakage. Thus one of the key
processes in salinity tolerance is to prevent lipid peroxidation to maintain the membrane
integrity [57, 62]. The oxidation of membrane lipids gives a reliable indication of an
extra free radical production leading to oxidative stress [63]. Data from this study
showed a lower MDA content in native-AMF inoculated plants. A similar reduction of
oxidative damage to lipids by AM symbiosis has been observed in tomato plants
subjected to salinity stress [23, 60]. These data, together with the lower electrolyte
leakage in maize plants inoculated with native AMF from Cabo de Gata, provides
evidence that the presence of native-AM symbiosis can prevent cell membrane injury.
This is in accordance to some other works that showed lower lipid peroxidation and
membrane permeability in AM plants compared to non-mycorrhizal plants [23, 57, 62,
64]. For instance, Kaya et al. [65] also reported that the electrolyte leakage in leaves of
Capsicum annum treated with 50 mM and 100 mM NaCl were decreased significantly
by AM inoculation. Again, plants inoculated with native AMF Ri CdG and Sc CdG had
lower electrolyte leakage than non-mycorrhizal plants or plants inoculated with Ri
collect. Several works have shown that AMF isolated from a saline area exhibited
significant higher symbiotic efficiency than non-saline AMF [19, 29]. Moreover,
Querejeta et al. [27] suggested that modulation of leaf gas exchange parameters in
Mediterranean environments by native-adapted AMF is of critical importance for host
plant performance in semiarid environments.
As discussed previously, the lower MDA concentrations in AM-inoculated
plants may have been due to the better performance of photosystem II and lower
generation of ROS in these plants. However, the activation of antioxidant enzymes such
as superoxide dismutase (SOD) or catalase (CAT) may have also accounted [14, 66].
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Our results showed that AM symbiosis significantly influenced SOD and CAT activities
to different extent depending on the AMF species, which might be the result of a
complex interaction among AMF, maize and the salinity stress. The reactive oxyradical
scavenging enzyme is an antioxidant system that plays a role maintaining cell
membrane stability in plant cell [67]. SOD enzyme catalyses the conversion of free O2•−
to O2 and H2O2. In the present work, the effect of salt stress on antioxidant enzyme
activity in maize revealed that the increase of SOD activity was higher in roots than in
shoots. In the root tissues, at 100 mM NaCl, all native AM-inoculated plants showed
significantly higher SOD activity and Ce CdG had the highest value. Several studies
reported that AMF enhance SOD activity in mycorrhizal plants under salt stress
conditions [57, 60, 68]. The greater SOD activity that we observed in mycorrhizal
plants could increase the capacity to scavenge superoxide radicals. Enhancement of
SOD activity under salt stress after the inoculation with salinity adapted fungi suggests
that native AMF improve the capacity of maize plants to cope with oxidative stress. In a
previous work, we provided evidence that the encoding gene for SOD in the fungus Ri
CdG was up-regulated under saline conditions. Hence we confirmed a fungal response
to the oxidative stress induced by salinity [29] which may have accounted also for the
reduced oxidative damage in the inoculated maize plants. Once the free O2•− is
detoxified by SOD activity, there is a need to scavenge hydrogen peroxide which is still
toxic and must be eliminated by conversion to H2O in subsequent reactions. A number
of enzymes regulate H2O2 intracellular levels in plants, and CAT is among the most
important ones [13]. Several authors have reported an enhancement of CAT activity by
inoculation with AMF [57, 60, 69]. However, in our work the effect of AMF on CAT
activity under salinity stress was little evident, which is in agreement with other reports
indicating that AM symbiosis did not affect CAT activity of salt stressed plants [23, 68].
The effects of AM symbiosis on the antioxidant systems observed in this study
were more prominent under the highest salt stress level (100 mM NaCl). The latter
suggests that under mild salts in the soil, maize antioxidant system could be enough to
cope with the stress. However, at higher levels of salt in the soil, AMF, particularly
salinity-adapted ones, are important to improve the capacity of maize to grow under
such unfavourable conditions. Greater SOD activity in plants inoculated with native
AMF compared with non-mycorrhizal or Ri collect plants was associated with lower
accumulation of H2O2 and less lipid peroxidation, indicating lower oxidative and
membrane damage in the native AMF-inoculated plants. Our observations are in
agreement with Bartels [70] that proposed both the prevention of oxidative stress and
the elimination of ROS as the most effective approaches used by plants to gain
tolerance against several abiotic stresses, including salinity.
Our results supports that AM inoculation enhances maize salt tolerance by
alleviating the salt induced oxidative stress and membrane damage. To this effect, the
better performance of photosystem II and stomatal conductance of plants inoculated
with the three native AMF must also have contributed through reduced ROS generation.
169
Chapter 5
This work demonstrates that AMF isolated from saline areas appear to be
physiologically adapted to the stress conditions, conferring higher salinity tolerance and
improving the growth of an important crop plant such as maize under salinity stress. In
agricultural lands with high levels of disturbance, inoculation is beneficial due to low
AM fungal inoculum potential [71]. Thus, from this work we highlight the potential use
of AMF from saline habitats to make successful inocula to improve crop growth and
yield in saline soils.
Acknowledgements
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CAPÍTULO 6
CHAPTER 6
Resumen
Antecedentes y objetivos:
Los ecosistemas mediterráneos afectados por salinidad se presentan ante un problema
creciente de degradación. La restauración biológica de los ecosistemas salinos se podría
lograr mediante el uso de especies de plantas halófitas junto con la inoculación de
hongos MA adaptados. Una planta de interés para su uso en la restauración de los
ambientes salinos, Asteriscus maritimus es altamente micotrófica. El objetivo del este
estudio fue evaluar la efectividad de hongos MA nativos y alóctonos en el
establecimiento y crecimiento de la halófita A. maritimus bajo condiciones de salinidad.
Métodos:
Se estudió la efectividad simbióticade cuatro aislados de hongos MA (tres nativos de un
suelo salino y uno alóctono de colección) en A. maritimus sometido a niveles crecientes
de salinidad. Se midieron parámetros fisiológicos de la planta en los que los hongos MA
pueden aliviar el efecto negativo del estrés salino, como la conductancia estomática y la
eficiencia del fotosistema II, acumulación de compuestos antioxidantes, actividad de
enzimas antioxidantes o daño oxidativo a lípidos.
Resultados:
Las plantas de A. maritimus mostraron una gran dependencia micorrícica incluso en
ausencia de estrés salino. Al nivel más alto de salinidad, las plantas inoculadas con
hongos nativos MA tuvieron mayor biomasa aérea, eficiencia del fotosistema II,
conductancia estomática, acumulación de glutatión y actividad de varias enzimas
antioxidantes que plantas inoculadas con el hongo MA de colección. Además, a este
nivel de sal, únicamente 30% de las plantas de A. maritimus inoculadas con el hongo
MA de colección sobrevivieron, mientras que en las plantas colonizadas por los hongos
nativos el porcentaje de supervivencia fue del 100%.
Conclusiones:
El presente estudio muestra la importancia de la inoculación con hongos nativos en
establecimiento, supervivencia y crecimiento de A. maritimus. El uso de un inóculo
adecuado de hongos MA nativos puede ser un elemento crucial para el éxito en la
recuperación de áreas salinas degradadas.
179
180
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181
Chapter 6
Abstract
Introduction
182
Chapter 6
183
Chapter 6
the Asteraceae family and known since long time to be highly mycotrophic (Mason
1928). In fact native plants thrive in various abiotically stressed ecosystems thanks to
AMF that have co-evolved and are essential for their adaptation to stressed conditions
(Rodriguez and Redman 2008). Nevertheless, in arid and semiarid ecosystems there is a
low density of AM propagules making difficult the successful reestablishment of native
plants (Jeffries et al. 2002). Thus, it is very important the ecophysiological study of
autochthonous AMF isolates and the knowledge of their mechanisms of salinity stress
adaptation and tolerance (Enkhtuya et al. 2000) in order to select the most adapted and
efficient species/strains of AMF to serve as inocula in revegetation programs (Ferrol et
al. 2004). Both halophytes and indigenous AMF isolates from saline habitats have
developed a variety of modifications to survive in saline environments, such as
regulating ionic homeostasis and detoxifying ROS (Zhu 2001; Ruiz-Lozano et al. 2012).
The biological restoration of saline ecosystems could be achieved by using halophytes
together with inoculation of adapted AMF. However the biochemical mechanisms by
which AM-colonized halophytic plants tolerate or reduce salt stress are poorly
understood.
In the present work, we studied the symbiotic effectiveness of four AMF strains
(three native isolates from a saline soil and one allochthonous, belonging to the EEZ
collection) in Asteriscus maritimus subjected to salinity stress. The native strains of
AMF were successfully isolated from the rizhosphere of A. maritimus at Cabo de Gata
Natural Park (Almería, SE Spain), which is the most arid ecosystem in Europe (Geiger
1973) with important salinity problems as well. The aim was to assess the effectiveness
of AM association among different AM isolates under saline conditions, to enhance the
establishment and growth of the halophyte A. maritimus for revegetation in salt affected
areas. In addition, we tried to elucidate the physiological and biochemical basis of the
mechanisms involved in the variability of the symbiotic performance.
Identification of the mycorrhizal strains isolated from Cabo de Gata Natural Park
AMF spores were separated from the soil samples by a wet sieving process
(Sieverding 1991). The morphological spore characteristics and their subcellular
structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-
glycerine (PVLG) (Koske and Tessier 1983); a mixture of PVLG and Melzer’s reagent
(Brundrett et al. 1994); a mixture of lactic acid to water at 1:1; Melzer’s reagent; and
water (Spain 1990). For identification of the AMF species, spores were then examined
using a compound microscope at up to 400-fold magnification as described for
glomeromycotean classification by Oehl et al. (2011). The species were identified based
on its spore morphology as a Rhizophagus intraradices (Schenk and Smith 1982),
184
Chapter 6
Experimental design
185
Chapter 6
Loamy soil was collected from Cabo de Gata Natural Park (Almería province)
(Spain, 36º45´24´´N 02º13´17´´W), sieved (5 mm), diluted with quartz-sand (<2 mm)
(1:1, soil:sand, v/v) and sterilized by steaming (100ºC for 1 h on 3 consecutive days).
The original soil had a pH of 8.7 [measured in water 1:5 (w/v)]; 0.26 % organic matter,
nutrient concentrations (g kg-1): N, 0.3; available P, 47.0 and soil electrical conductivity
3.95 dS m-1.
Seeds of Asteriscus maritimus. L were sown on a vermiculite:sand mixture (1:1,
v/v) for germination. Two weeks after germination, four seedlings of A. maritimus were
transplanted per pot containing 900 g of the same soil/sand mixture as described above
and thinned to one plant per pot after establishment was successfully done (plants were
selected with uniform size for each treatment).
Inoculation treatm
Growth conditions
The experiment was carried out under glasshouse conditions with temperatures
ranging from 19 to 25ºC, 16/8 light/dark period, and a relative humidity of 50-60%. A
photosynthetic photon flux density of 800 μE m-2 s-1 was measured with a light meter
(LICOR, Lincoln, NE, USA, model LI-188B). Water was supplied daily to the entire
period of plant growth to avoid any drought effect. Plants were established for 6 weeks
prior to salinization to allow adequate plant growth and symbiotic establishment. Three
concentrations (0, 100, and 175 mM NaCl) of saline solution were reached in the soil
substrate by adding appropriate dilutions of a stock 2 M saline solution. The
concentration of NaCl in the soil was increased gradually on alternative days to avoid an
osmotic shock. It took 6 weeks, to reach the desired 100 and 175 mM NaCl levels. The
electrical conductivities in the soil:sand mixture used as growing substrate were 2.2, 8.9
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Chapter 6
and 13.5 dS m-1 for the salt levels of 0, 100, and 175 mM NaCl, respectively. Plants
were maintained under these conditions for additional 8 weeks.
Symbiotic development
Biomass production
At harvest (5 months after planting), the shoot and root system were separated
and the shoot dry weight (SDW) and root dry weight (RDW) were measured after
drying in a forced hot-air oven at 70ºC for two days. Ten plants per treatment were used
(except for plants inoculated with Ri collect and subjected to 175 mM NaCl, where only
three plants survived).
Photosynthetic efficiency
Stomatal conductance
Stomatal conductance was measured two hours after light turned on by using a
porometer system (Porometer AP4, Delta-T Devices Ltd, Cambridge, UK) following
the user manual instructions. Stomatal conductance measurements were taken in the
third youngest leaf from five different plants from each treatment.
Proline content
Free proline was extracted from 0.5 g of fresh leaves and roots (Bligh and Dyer
1959) in five plants per treatment. The methanolic phase was used for quantification of
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Chapter 6
Glutathione content
Ascorbate content
Lipid peroxides were extracted by grinding 500 mg of leaves and roots of five
plants from each treatment with an ice-cold mortar and 6 ml of 100 mM potassium
phosphate buffer (pH 7). Homogenates were filtered through one Miracloth layer and
centrifuged at 15,000 × g for 20 min. The chromogen was formed by mixing 200 μl of
supernatants with 1 ml of a reaction mixture containing 15% (w/v), trichloroacetic acid,
0.375% (w/v) 2-thiobarbituric acid (TBA), 0.1% (w/v) butyl hydroxytoluene, 0.25N
HCl and then incubating the mixture at 100ºC for 30 min (Minotti and Aust 1987). After
cooling at room temperature, tubes were centrifuged at 800 × g for 5 min and the
supernatant was used for spectrophotometric reading at 532 nm. Lipid peroxidation was
estimated as the content of 2-thiobarbituric acid-reactive substances (TBARS) and
expressed as equivalents of malondialdehyde (MDA) according to Halliwell and
Gutteridge (1989). The calibration curve was made using MDA in the range of 0.1-10
188
Chapter 6
nmol. A blank for all samples was prepared by replacing the sample with extraction
medium, and controls for each sample were prepared by replacing TBA with 0.25N
HCl. In all cases, 0.1% (w/v) butyl hydroxytoluene was included in the reaction
mixtures to prevent artifactual formation of TBARS during the acid-heating step of the
assay.
Enzyme extraction was done as described before by Aroca et al. (2001) with
slight modifications. Five hundred mg of leaf and root fresh tissues were homogenized
separately in a cold mortar with 4 ml of 100 mM phosphate buffer (pH 7.0) containing
0.1 mM DTPA (diethylenetriamine pentaacetic acid; a metal chelating agent) and 40 mg
PVPP (polyvinylpolypyrrolidone), which removes phenolics and alkaloids from plant
extracts, avoiding interference with spectrophotometric measurements and enhancing
enzyme stability. The homogenate was centrifuged at 20,000 x g for 20 min at 4ºC. The
supernatant was separated and used to determine the activity of antioxidant enzymes.
All enzymatic activities were measured in a spectrophotometer InfiniteR 200 PRO series
(Tecan Trading AG, Switzerland) at 25ºC. Total protein was assayed by the method
described by Bradford (1976).
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Chapter 6
Statistical Analysis
Statistical analysis was performed using SPSS 19.0 statistical program (SPSS
Inc., Chicago, IL, USA) performing first a one-way ANOVA followed by the Tukey
test with P< 0.05 as the significance cut-off. Two independent statistical analyses were
carried out: the first to analyze data from the different AMF treatments within each
saline level and the second one to analyze data from each fungal species at increasing
salinity.
Results
Symbiotic development
All the fungi exhibited similar colonization rates at all salinity levels (Table 1).
Within each salinity level, the highest rate of AM root colonization was achieved in
plants inoculated with Ri collect (up to 77%). High levels of root colonization were also
found in plants inoculated with Ri CdG and Sc CdG (Table 1). In contrast, the lowest
root colonization was always found in plants colonized by Ce CdG (about 25%).
Non-mycorrhizal A. maritimus plants did not survive under the conditions of this
experiment, even in the absence of salinity. At the highest salinity level, only 30% of
plants inoculated with Ri collect survived. The rest of the inoculation treatments had
100 % of survival rate (Table 1).
The increase of salt application affected negatively the shoot biomass production
in all treatments except in plants inoculated with Ri CdG (Table 1). In plants inoculated
with Sc CdG and Ce CdG the decrease was only observed at 100 mM. In plant
inoculated with Ri collect the reduction was not significant until 175 mM NaCl was
reached (Table 1). However, under the highest saline conditions, shoot dry weight was
higher in plants colonized by AMF from Cabo de Gata than in those colonized by Ri
collect (Table 1). In contrast, the root biomass production decreased at each salinity
level in plants inoculated with Ri both collect and CdG, two of them showing the lowest
values of root dry weight at 175 mM NaCl (Table 1). Root dry weight was unaffected in
plants inoculated with Ce CdG. Root dry weight was reduced to a half in plants
inoculated with Sc CdG at 100 mM NaCl and it was not changed further at 175 mM
NaCl, being the highest value at this salt level (Table 1).
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Chapter 6
Table 1. Percentage of mycorrhizal root colonization, percentage of survival, shoot and root dry weight
(g plant-1) in Asteriscus maritimus plants. NM represents non-mycorrhizal control plants; Ri collect,
plants inoculated with the collection Rhizophagus intraradices strain; Ri CdG, plants inoculated with the
native Rh. intraradices CdG strain; Sc CdG, plants inoculated with the native Septoglomus claroideum
CdG strain and Ce CdG plants inoculated with the native Claroideoglomus etunicatum CdG strain. Plants
were subjected to 0, 100 or 175 mM NaCl. Different letters indicate significant differences (p < 0.05)
among fungal treatments at each salt level (a, b, c, d) or among salt levels for each AMF treatment: Ri
collect (A, B, C), Ri CdG (D, E, F), Sc CdG (G, H, I) or Ce CdG (J, K, L).
0 0 0 0 0
NM 100 0 0 0 0
175 0 0 0 0
Photosystem II efficiency
Salinity application did not cause any significant descent in photosynthetic
efficiency in A. maritimus plants inoculated with the three native AMF from Cabo de
Gata (Fig. 1A). Only plants inoculated with Ri collect decreased the efficiency of
photosystem II when they were subjected to 175 mM NaCl (Fig. 1A).
Stomatal conductance
191
Chapter 6
0.7 A D G J A
Photosynthetic efficiency
G D
a D a J G J
a a a a a A
a a a a
0.6
0.5 B
b
0.4
0.3
0 mM 100 mM 175 mM
180 Ri collect
J
Ri CdG B
160 G a Sc CdG
D
Stomatal conductance (mmolH2Om-2s-1)
A a Ce CdG
140 a
a
120
100
80
H
25 B E H K K
E a
a a a a B a
a
20
b
15
10
5
0
0 mM 100 mM 175 mM
Fig. 1. Efficiency of photosystem II (A) and stomatal conductance (mmol H2O m-2s-1) (B) in Asteriscus
maritimus plants. Black bars represent plants inoculated with the collection Rhizophagus intraradices
strain (Ri collect); white bars, plants inoculated with the native Rh. intraradices CdG strain (Ri CdG);
grey bars, plants inoculated with the native Septoglomus claroideum CdG strain (Sc CdG) and lined bars,
plants inoculated with the native Claroideoglomus etunicatum CdG strain (Ce CdG). Plants were
subjected to 0, 100 or 175 mM NaCl. Different letters indicate significant differences (p < 0.05) among
fungal treatments at each salt level (a, b, c) or among salt levels for each AMF treatment: Ri collect plants
(A, B, C), Ri CdG (D, E, F), Sc CdG (G, H, I) or Ce CdG (J, K, L).
Accumulation of proline
The accumulation of proline was more pronounced in shoot than in root tissues
(Fig. 2A,B). In leaves proline increased after exposure to salinity in the growth medium.
Differences between treatments were only evident at 100 mM NaCl, where plants
inoculated by any of the two Ri strains had the highest values (Fig. 2A). In roots only
plants inoculated with Ri CdG increased the accumulation of proline with increasing
salinity. All the other fungi increased the accumulation of proline at 100 mM and did
not change it at 175 mM NaCl (Fig. 2B). A. maritimus inoculated with Ri CdG or Ce
CdG had the lowest proline value at 100 mM NaCl(Fig. 2B).
192
Chapter 6
20 A D G J
B
E a a a a A
a
a
15
K
H
b
10 b
Proline content (μmol g-1 DW)
Ri collect
5 I L Ri CdG
C F Sc CdG
a a a a Ce CdG
0
0 mM 100 mM 175 mM
A D
A
G G J
14 a a
a E
ab
J a a B
12 b
b
10
8
6
4
B F H K
2 a a a a
0
0 mM 100 mM 175 mM
Fig. 2. Shoot (A) and root (B) proline content in A. maritimus plants. See legend for Fig. 1.
Glutathione content
Ascorbate content
In leaves, the content of ascorbate due to the increasing salinity only rose in Ri
CdG-inoculated plants at 100 mM NaCl and in plants colonized by Ri collect when
subjected to 175 mM NaCl (Fig. 4A). At 100 mM NaCl plant inoculated with Ce CdG
showed the lowest ascorbate content but at the higher level of NaCl no differences were
observed. In roots, exposure to 100 or 175 mM NaCl did not significantly affect the
ascorbate content in A. maritimus plants (Fig. 4B). But again, at 100 mM NaCl
treatment, Sc CdG roots showed the highest ascorbate values. At 175 mM NaCl the
highest values were found in Ri collect roots instead.
193
Chapter 6
120 J
D G a A
100 A GH
A D a a
a E ab K
80
K ab a
Glutathione content (nmol g-1 DW)
60 a b B
H ab b Ri collect
40 b Ri CdG
20 Sc CdG
Ce CdG
0
0 mM 100 mM 175 mM
G
80 a
A D D B
60 H H J
D a a AB b
B a a J ab J
b b
40 a a b
20
0
0 mM 100 mM 175 mM
Fig. 3. Shoot (A) and root (B) glutathione content in A. maritimus plants. See legend for Fig. 1.
G G
16 a D A G
a
J ab a J A
14 B B DE a
12 ab a
ab E bc J
a
10 b
c
8
Ascorbate content (mmol g-1 DW)
6 Ri collect
4 Ri CdG
Sc CdG
2 Ce CdG
0
0 mM 100 mM 175 mM
A
10 A J G a
a D a B
a
8 D GH ab D J
Ri collect
a a B J b H ab Ri CdG
6 b b Sc CdG
b
Ce CdG
4
0
0 mM 100 mM 175 mM
Fig. 4. Shoot (A) and root (B) ascorbate content in A. maritimus plants. See legend for Fig. 1.
194
Chapter 6
A A
30 a D a D
a J A
25 E a
J
Oxidative damage to lipids (nmol MDA g-1 DW)
a
20 a b
B
K G
15 ab
H bc b
H
10 c Ri collect
c Ri CdG
5 Sc CdG
Ce CdG
0
0 mM 100 mM 175 mM
30 A
a A D A B
25 a
D a ab D J
20 J a
b J a
b
15 b G
10 H b
I c
5
c
0
0 mM 100 mM 175 mM
Fig. 5. Shoot (A) and root (B) oxidative damage to lipids in A. maritimus plants. See legend for Fig. 1.
195
Chapter 6
the lowest activity at 0 and 100 mM NaCl, together with Ri collect-inoculated plants
(Fig. 6A). At 175 mM NaCl plants inoculated with Sc CdG had the highest SOD
activity, followed by those inoculated with Ce CdG. Root SOD activity increased by
salt application in plants colonized by Sc CdG (Fig. 6B). Ri CdG inoculated plants
showed the lowest SOD activity under non-saline conditions. No significant differences
in root SOD activity among inoculation treatments were observed at 100 mM NaCl
(Fig. 6B).
G
6 Ri collect a
Ri CdG A
5 Sc CdG J
Ce CdG H
4 b
a
D
3 A H D
SOD activity (U min-1mg-1prot)
a
a D a c
2
a B
K K
1 b
b b
nd
0
0 mM 100 mM 175 mM
G
20
a
B
15
A
A H J
10 a D H J
a a a D J
a a a
E b b
5
b
nd
0
0 mM 100 mM 175 mM
Fig. 6. Shoot (A) and root (B) SOD activity in A. maritimus plants. See legend for Fig. 1. (nd means non-
determined enzyme activity).
196
Chapter 6
D
40 Ri collect a
35 Ri CdG G A
Sc CdG a G J
30 Ce CdG
b b
25 H K
GR activity (nmol NADPH min-1mg-1prot) K
20 A E a a A E
b
15 a a b b
10
5
nd
0
0 mM 100 mM 175 mM
70 G
a B
60
50 H
D
40 a
I b
30 A
A E a J
b E JK
20 ab ab K b c
b
10 b
nd
0
0 mM 100 mM 175 mM
Fig. 7. Shoot (A) and root (B) GR activity in A. maritimus plants. See legend for Fig. 1. (nd means non-
determined enzyme activity).
A
60 a
Ri collect
Ri CdG A
50 Sc CdG
Ce CdG G
40 b D G
APX activity (nmol Asc min-1mg-1prot)
30 a a
J
20 E b
10 B E H K c K
a a a a
nd
c
0
0 mM 100 mM 175 mM
G
400 a
A B
350 H H
a J
300 B a ab D J
D ab
250 a b b
b
200 E K
150 b b
100
50
nd
0
0 mM 100 mM 175 mM
Fig. 8. Shoot (A) and root (B) APX activity in A. maritimus plants. See legend for Fig. 1. (nd means non-
determined enzyme activity).
197
Chapter 6
Discussion
198
Chapter 6
efficiency with A. maritimus, as well as a 100% of survival rate under 175 mM NaCl. In
fact, the high percentage of root colonization by Ri collect could demand excessive
carbohydrates from the plant (Klironomos 2003).
In this study, when plants of A. maritimus were subjected to high salinity stress
(175 mM NaCl), plants inoculated with the three native-AMF exhibited better
performance of photosystem II and higher stomatal conductance. Results on efficiency
of photosystem II and stomatal conductance indicate that plants inoculated with native-
AMF may improve the net assimilation rates by protecting the photosynthetic
machinery and enhancing transpiration rates. Some other authors showed a similar
tendency (Sheng et al. 2008; Hajiboland et al. 2010) and Estrada et al. (2012) reported
that the improvement in photosystem II was higher with native AMF than with
collection fungi together with the enhancement of plant stomatal conductance, in
agreement with Querejeta et al. (2006). These two effects may have accounted for the
enhanced growth of A. maritimus during high salinity stress.
In addition to the beneficial effect of native mycorrhizal colonization in A.
maritimus salt tolerance by improving photosynthetic ability and stomatal conductance;
water and nutrient uptake, ion balance and osmolite concentration among others may
have also contributed to it (Ruiz-Lozano et al. 2012). It has been previously shown that
a native AMF from Cabo de Gata developed better under salinity than a collection
fungal strain (Estrada et al., 2012). Thus, the extensive hyphal network of native AMF
can explore a larger soil volume, contributing to water and nutrient uptake (Evelin et al.
2012). The maintenance of proper ionic homoeostasis and osmotic adjustment may also
prevent salt injury. The synthesis of organic osmolites, especially proline that is
normally located in the cytosol, appears to be of importance in osmotic adjustment
(Moghaieb et al. 2004). Our results did not show remarkable differences in proline
content among fungal treatments, although proline accumulation increased in all
treatments with the salinity stress. The latter may be explained as halophytic plants are
themselves tolerant to salinity in part because they are able to maintain a high osmotic
potential through the accumulation of organic solutes (Bradley and Morris 1991).
Salinity stress leads to secondary oxidative damage, thus the improvement of
stress tolerance is often related to enhancement of contents of antioxidant compounds in
plants. Due to the toxicity of ROS, plants have developed appropriate detoxification
systems to remove these molecules. These systems include, among others, non-
enzymatic antioxidants soluble compounds, such as glutathione and ascorbate that are
major plant metabolites that regulate essential cell functions and play a key role in
antioxidant defence (Tunc-Ozdemir et al. 2009). Glutathione reacts with superoxide
radicals, peroxy radicals and singlet oxygen and forms oxidized glutathione and other
disulphides (Meyer 2007). The ascorbate is involved in the removal of H2O2 by
ascorbate peroxidases, which use ascorbate as electron donor, and is closely related to
glutathione in the ascorbate-glutathione cycle where glutathione participates in the
reduction of oxidized ascorbate (Noctor and Foyer 1998). Our results showed that
199
Chapter 6
glutathione content had the most significant differences among treatments. A. maritimus
plants inoculated with native AMF increased it shoot glutathione content with
increasing salinity. Plants inoculated with Ri collect reduced the content at 175 mM
NaCl and at this salinity level, the content of glutathione in shoots was lower compared
to native AMF-inoculated plants. In contrast, the ascorbate content did not show a
significant trend. From these results we may propose that A. maritimus has preference
for glutathione as an antioxidant compound compared to ascorbate. The reason for that
could be related to the additional physiological functions ascribed to glutathione, such
as the induction of enzyme activities and its participation in sulphur metabolism and
regulation of gene expression (Foyer et al. 1995).
Another major effect of salinity stress in plants is the loss of membrane integrity
due to the oxidation of membrane lipids. Thus the prevention of lipid peroxidation is of
crucial importance to maintain membrane integrity (Garg and Manchanda 2009). Our
findings showed that in A. maritimus, MDA content (a specific product of lipid
peroxidation induced by ROS) increased with salinity in shoots but not in roots, with
exception of Sc CdG inoculated plants, which always increased the MDA content. Lipid
peroxidation is considered an useful indicator of cellular oxidative damage (Li et al.
2012), however it might not be a good indicator in halophytes due to the particular
characteristics of these plants. We cannot compare with some other works that observed
a reduction of oxidative damage to lipids by AM symbiosis in plants subjected to salt
stress (He et al. 2007; Hajiboland et al. 2010) because of the lack of non-mycorrhizal
plants. In spite of that, we observed that, at each salt level, plants inoculated with Sc
CdG always had the lowest MDA content.
Oxidative damage in plants can be also ameliorated by an efficient antioxidative
enzyme system that may confer plants the ability to tolerate salt stress (Mittler 2002).
The induction of ROS-scavenging enzymes, such as SOD, GR and APX is the most
common mechanism for detoxifying ROS synthesized during stress response
(Hajiboland et al. 2010). In halophytes the effects of AMF on the activities of
antioxidant enzymes have been rarely studied (Li et al. 2012). Data from this study is
difficult to discuss because there are no data with respect of Ri collect inoculated plants
at 175 mM NaCl. Despite of all the above mentioned, the activity of SOD in shoots only
increased in plants inoculated with Sc CdG and Ce CdG. In roots, plants inoculated with
Sc CdG showed the most important increase in SOD activity, having the highest activity
both in shoots and in roots. This may have accounted for the lower MDA content in A.
maritimus plants inoculated with Sc CdG compared to the rest of the treatments.
Although the pattern of GR activity in roots was not relevant, in shoots of A.
maritimus it was higher in plants inoculated with the three native AMF at the highest
level of salinity. The enhanced GR activity in native AMF inoculated plants may have
contributed to generate reduced glutathione that plays a pivotal role in the ascorbate-
glutathione cycle among others. As for the APX activity, the data showed that it was
enhanced by the increase in salinity in all treatments both in shoots and in roots. In
200
Chapter 6
accordance to He et al. (2007), our results indicated that AMF induce APX activity.
Nonetheless, native AMF did not enhance the activity more than the Ri collect fungus
contrary to the other studied enzymes. It is known that the response of each enzyme
differs with respect not only to the fungal species but to the host plant (Roldán et al.
2008; Evelin et al. 2009; Ruiz-Lozano et al. 2012).
Summarizing, the present study showed the elevated degree of mycotrophy of A.
maritimus plants, especially regarding native AMF from its environment. In addition,
we have observed several physiological mechanisms by which native AMF ameliorate
the detrimental effects of salinity stress in A. maritimus better than an exotic AMF, such
as better photosynthetic efficiency or higher levels of glutathione and of certain
antioxidant enzyme activities. In degraded semiarid Mediterranean areas, Alguacil et al.
(2011) demonstrated the important impact of inoculation with native AM fungi on the
growth of shrub species. Moreover, reestablishment of plants species and restoration of
specific ecosystems need careful considerations regarding the AMF inocula and the
plant species used. Also the introduction of novel AMF species or isolates to the site
could result in the out-competition of native fungal strains that could provoke the up-
coming or survival of invasive plant species (Schwartz et al. 2006). Thus, inadequate
inocula should be avoided due to possible negative effects on plant species and the plant
community. Van der Heijden et al. (1998) showed that under particular environmental
conditions, different AMF strains had negative or positive effect on the colonized plant,
pointing out the importance of the interactions between AMF and plant species. The
results in the present work remark the importance of native AMF inoculation in A.
maritimus establishment, survival and growth for successful restoration of
Mediterranean areas, particularly on sites with high salt levels in the soil.
Acknowledgements
201
Chapter 6
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CONCLUSIONES
CONCLUSIONS
Conclusiones
CONCLUSIONES
2. En las dunas del Parque Natural de Cabo de Gata se ha descrito una nueva
especie de MA asociada a la rizosfera de Asteriscus maritimus y existe la
posibilidad de encontrar más especies aún desconocidas.
6. Las plantas de maíz inoculadas con los tres hongos nativos de Cabo de Gata
mostraron mayor eficiencia del fotosistema II y conductancia estomática, lo que
habría contribuido a reducir la fotorrespiración y la producción de ROS.
Además, los sistemas antioxidantes de estas plantas fueron inducidos en mayor
medida que en las plantas no micorrizadas o en las inoculadas con el hongo MA
de colección, siendo el daño oxidativo significativamente menor.
211
Conclusiones
8. Existen diferencias en los mecanismos por los que los hongos MA nativos de
Cabo de Gata incrementan la tolerancia de la planta hospedadora a la salinidad.
Esto estaría modulado, igualmente, por las características fisiológicas de la
especie de planta utilizada.
212
Conclusions
CONCLUSIONS
3. The native isolate from Cabo de Gata Glomus (= Rhizophagus) intraradices has
shown to have greater development capacity under saline conditions than an
isolate of the same species from collection, indicating higher adaptation to
salinity than the fungus from collection. This effect may be related to the
significant up-regulation in the expression of the genes GintBIP, Gint14-3-3 and
GintAQP1, in the fungus isolated from Cabo de Gata.
4. Under saline conditions, the native isolate from Cabo de Gata G. intraradices
had also higher symbiotic efficiency with maize plants than an isolate of the
same species from collection, promoting a greater development of the plants.
5. The use of native AM fungi isolated from Cabo de Gata increased the salt
tolerance of a glycophyte plant of agronomic interest, such as maize, more than
a fungus from collection. Plants inoculated with the three native fungi showed a
significant increase of the K+/Na+ ratios in their tissues. This effect was
correlated with the regulation of expression of ZmAKT2, ZmSKOR and ZmSOS1
genes in the roots of such plants, contributing to improve the ionic homeostasis.
6. Maize plants inoculated with the three native AM fungi from Cabo de Gata
showed higher efficiency of photosystem II and stomatal conductance, which
should have contributed to reduce photorespiration and ROS production. In
addition, the antioxidant systems of these plants were induced to a greater extent
than in non-mycorrhizal plants or plants inoculated with the fungus from
collection, and they had a significantly lower oxidative damage.
213
Conclusions
8. There are differences in the mechanisms by which native AMF of Cabo de Gata
increase host plant tolerance to salinity. This would be also modulated by the
particular physiological characteristics of the plant species used.
214